Investigation of the molecular biology underlying the pronounced high gene targeting frequency at the Myh9 gene locus in mouse embryonic stem cells.

The generation of genetically modified mouse models derived from gene targeting (GT) in mouse embryonic stem (ES) cells (mESCs) has greatly advanced both basic and clinical research. Our previous finding that gene targeting at the Myh9 exon2 site in mESCs has a pronounced high homologous recombination (HR) efficiency (>90%) has facilitated the generation of a series of nonmuscle myosin II (NM II) related mouse models. Furthermore, the Myh9 gene locus has been well demonstrated to be a new safe harbor for site-specific insertion of other exogenous genes. In the current study, we intend to investigate the molecular biology underlying for this high HR efficiency from other aspects. Our results confirmed some previously characterized properties and revealed some unreported observations: 1) The comparison and analysis of the targeting events occurring at the Myh9 and several widely used loci for targeting transgenesis, including ColA1, HPRT, ROSA26, and the sequences utilized for generating these targeting constructs, indicated that a total length about 6 kb with approximate 50% GC-content of the 5' and 3' homologous arms, may facilitate a better performance in terms of GT efficiency. 2) Despite increasing the length of the homologous arms, shifting the targeting site from the Myh9 exon2, to intron2, or exon3 led to a gradually reduced GT frequency (91.7, 71.8 and 50.0%, respectively). This finding provides the first evidence that the HR frequency may also be associated with the targeting site even in the same locus. Meanwhile, the decreased trend of the GT efficiency at these targeting sites was consistent with the reduced percentage of simple sequence repeat (SSR) and short interspersed nuclear elements (SINEs) in the sequences for generating the targeting constructs, suggesting the potential effects of these DNA elements on GT efficiency; 3) Our series of targeting experiments and analyses with truncated 5' and 3' arms at the Myh9 exon2 site demonstrated that GT efficiency positively correlates with the total length of the homologous arms (R = 0.7256, p<0.01), confirmed that a 2:1 ratio of the length, a 50% GC-content and the higher amount of SINEs for the 5' and 3' arms may benefit for appreciable GT frequency. Though more investigations are required, the Myh9 gene locus appears to be an ideal location for identifying HR-related cis and trans factors, which in turn provide mechanistic insights and also facilitate the practical application of gene editing.

Introduction

Transgenic animals that have had their genomes modified by genetic manipulation are extensively used to investigate the in vivo biological functions of genes as well as to mimic human diseases [1-5]. The mouse is the most widely engineered and commonly used organism for biomedical research. Its embryonic stem (ES) cell-mediated gene targeting has been a critical technology for generating such genetically modified mice [6, 7]. Moreover, the homologous recombination (HR)-based insertion, deletion or point mutation of the genome in ES cells and the consequent generation of targeted gain- and loss-of-function alleles have allowed the creation of thousands of mouse models for different purposes [1, 8, 9]. It is well known that the event of HR-mediated modification is rare. Even in mouse ES cells, obtaining the desired ES clone has therefore been a critical but time-consuming and labor-intensive step in previous studies [3, 10, 11]. Numerous factors including the length of the homologous arms, and the structure of the targeting vector, the targeted loci, the utilization of isogenic DNA and the status of ES cells, can affect the HR efficiency have been investigated and described [12-16]. However, the HR efficiency varies greatly even when the general principles are complied with. Furthermore, the conclusions from investigating the above factors may not be always consistent. For instance, the length of targeting arms is thought to be an important determinant of targeting efficiency [17], while increasing the targeting arm length does not always lead to the elevation of HR frequency [14]. These suggest the mechanisms underlying these influencing factors still remain elusive.

With the recent advent of a series of programmable nucleases or genome editing tools including zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein9 (Cas9) (CRISPR/Cas9), the GT frequency in mammalian cells including mouse ES cells has been dramatically improved [18-23], suggesting the compatibility of artificial nucleases with traditional gene targeting methods. Therefore, these new technologies have stimulated scientific attention, since they have the potential of bypassing the requirement of using mouse ES cells in the generation of genetically engineered mice. However, these technologies are also accompanied by some disadvantages, in particular, the off-target effects and the inability to insert large DNA fragments [24], as further substantiated by a recent study [25]. In contrast, the time-consuming shortcoming of the traditional method has been overcome by a recent report, which demonstrated that the approach of gene targeting in mouse extended pluripotent stem (EPS) cells, coupled with tetraploid complementation technology, can generate mutant mice in approximately 2 months [26]. Additionally, targeted alterations of the genome in ES cells, created with or without the assistance of gene editing tools, are widely used for various purposes, such as the in vitro differentiation of ES cells and the screening of druggable chemicals [23]. These highlight the necessity to investigate the intrinsic properties of HR-based gene targeting in ES cells for further improving the efficiency and for providing valuable clues to apply gene targeting in other mammalian cells.

Our previous finding indicated that gene targeting at the exon2 site of the Myh9 gene which encodes the protein of nonmuscle myosin heavy chain IIA (NMHC IIA) has a pronounced high HR efficiency (>90%) in mouse ES cells. This unique property has been applied to the production of a series of nonmuscle myosin II (NM II) related mutant mouse lines (≥7) [27-30]. Moreover, this finding has also been extended to the site-specific insertion of other exogenous genes in mouse ES cells [31]. In the present study, we intended to investigate the molecular biology underlying this distinctive HR efficiency from multiple aspects.

Materials and methods

This work was approved by the Animal Ethics Committee of Hunan Agricultural University, Hunan, China. No animal experiments were conducted, no ethic permits were therefore needed for this report, which complied with all of the relevant regulations.

Bioinformatics analysis

A 3950 bp sequence immediately before and a 1327 bp sequence immediately after the Myh9 gene exon2 across various species (including mouse, human, chimpanzee, pig, rabbit, and rat) were retrieved from useast.ensembl.org and further confirmed with genome.ucsc.edu. The comparison and alignment of the sequence region (<6 kb) around the Myh9 gene exon2 across distinct species was performed by using Clustal W in the DNAStar version 7.10 software (Lasergene) [32]. The DNA features including GC-content, simple sequence repeat (SSR), short interspersed nuclear elements (SINEs), and long interspersed nuclear elements (LINEs), other DNA elements in the sequences used for creating targeting constructs were analyzed via Repeat Masker Web Server (http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker). The CpG islands in these sequences were determined using CpG report software (https://www.ebi.ac.uk/Tools/seqstats/emboss_newcpgreport/).

Generation of targeting constructs

The constructs targeting to the Myh9 gene exon2, intron2 and exon3 sites were separately generated by using the same strategy as described previously [27, 31]. Briefly, DNA fragments for the 5’ and 3’ homologous arms were amplified by PCR using the primer pairs listed in S1 Table and the template of 129/Sv genomic BAC clone DNA, and then cloned into the vector mpNTKV-LoxP described previously [27]. Likewise, the truncated 5’ and 3’ homologous arms were firstly amplified by PCR with the primer pairs listed in S1 Table and the template of 129/Sv genomic BAC clone DNA, and then various combinations of the 5’ and 3’ homologous arms were cloned into the mpNTKV-LoxP vector. Nucleotide sequences of the cloned DNA fragments were verified in all cases by sequencing. All targeting vectors were linearized by restriction enzymes before electroporation.

Culture and electroporation of mouse ES cells

The mouse V6.5 ES cells used in this study were originally derived by Eggan et al [33] from F1 hybrid mice with 50% C57BL/6 and 50% 129/Sv mixed genetic background (Notably, the sequences of the targeting constructs are isogenic to both chromosomes from C57BL/6 and 129/Sv genetic background in terms of the Myh9 gene region involved.), cultured and electroporated with linearized targeting vectors as described before [27, 29], followed by drug selection using 400μg/ml G418 and 200μM ganciclovir. Drug resistant colonies were picked and expanded for the preparation of genomic DNA.

PCR identification of recombination events

HR events occurring at the Myh9 gene exon2 (including those truncated targeting constructs), intron2 and exon3 sites were separately identified by PCR with the primer pair (the forward primer located at the neomycin, while the reverse primer resided immediately outside the 3’ short arm) listed in S1 Table. The preparation of genomic DNA, the components of PCR system and the PCR reaction conditions completely followed the previous description [31]. The PCR products were analyzed by electrophoresis on 1.0% agarose gel, the wild-type allele produced no band while the targeted allele yielded an expected ≥2.1, 2.2, 2.3 kb band, respectively, as indicated in S1 Table. At least five randomly selected PCR-amplified products were excised, mixed, extracted and cloned into the T-easy vector for sequencing (Promega). The sequencing results could further confirm the gene targeting recombinants.

Data analysis

The relationship between the length of homologous arms and the targeting efficiency, as well as the significance, were analyzed with GraphPad.Prism.v5.0 software (GraphPad Software inc, CA, USA).

Results and discussion

Bioinformatic analysis of the Myh9 gene locus and other widely used loci for site-specific integration of exogenous genes

As demonstrated in previous studies, gene targeting at the Myh9 gene locus, in particular the exon2 site, had a pronounced high HR efficiency (>90%) in mESCs [27, 29–31]. Although GT efficiency in mESCs is considered 10–100 fold higher than that in mammalian somatic cells [34]. We are not aware of any reports showing such a high GT efficiency in mESCs under similar experimental conditions. Thus, the Myh9 gene locus can be added to the list of a few identified mitotic HR hotspots [35-37]. This finding also evoked our further curiosity about the molecular biology underlying it. Furthermore, the mechanisms responsible for the higher relative rate of gene targeting in mESCs than those in somatic cells remain unclear. As an initial effort, we examined whether the Myh9 locus has some common genetic features with other widely used loci for site-specific integration of exogenous genes, including collagen alpha 1 (ColA1), hypoxanthine phosphoribosyltransferase (HPRT) and ROSA26. To this aim, we firstly collected and summarized targeting events occurring at these loci from the references, as indicated in Table 1. Though there were several differences in the length of 5’ and 3’ arms, the ratio of them, ES cell lines used, the selection markers utilized, appreciable targeting efficiencies were obtained in these loci. Without considering other factors, several characteristics could be observed: a) These loci have at least one unique property, such as only one copy of exogenous gene insertion at the HPRT locus, high expression of exogenous gene at the ColA1, Myh9 and ROSA26 loci, thereby being widely used for targeted transgenesis; b) Generally, a 5–6 kb total length of homologous arms was utilized, while longer total length of homologous arms did not mean higher targeting efficiency; c) It seemed that the length ratio of the 5’ and 3’ arms also matters, as further demonstrated in our subsequent experiments. Next, we examined whether there exist some common genetic features among these loci. For this purpose, the sequences used for generating the constructs targeting to these loci were analyzed as described in Materials and Methods, the analytical results based on the 5’ or 3’ arm were summarized in Table 2. As far as these sequences were concerned, several features could be generalized: 1) GC-content in these sequences for the 5’ and 3’ arms displayed a range of 38–68%, a similar and about 50% GC-content seemed better than other cases with regard to GT efficiency; 2) There existed different DNA elements in the 5’ or 3’ arm of these constructs, such as simple sequence repeat (SSR), short interspersed nucleotide elements (SINEs), long interspersed nucleotide elements (LINEs), even CpG island or other elements, the effects of these elements on targeting efficiency have also been suggested [38]; 3) Notably, different DNA elements were distributed in the 5’ and 3’ arms of them, no common genetic properties could therefore be identified from these sequences. Furthermore, the practical influences of these DNA elements on GT frequency remained to be substantiated.

Table 1

Comparison of the HR efficiency and features of frequently targeted loci/sites.

Locus/Gene	Total length of homologous arms (kb)	Length of 5’ arm (kb)	Length of 3’ arm (kb)	Ratio of the length of 5’ to 3’ arm	ES cell lines	Positive/Negative selection marker	Targeting efficiency (%)	References
Myh9	5.7	4.0	1.7	2.3:1	V6.5	Neomycin/TK	91.7	[31]
ColA1	6.0	3.3	2.7	1.2:1	V6.5	Neomycin/-	80.0	[39, 40]
HPRT	9.6	3.8	5.8	1:1.5	BPES	HAT/-	High while detailed information undisclosed	[39, 41–44]
ROSA26	5.4	1.1	4.3	1:3.9	AK7	Neomycin/DTA	34.8	[39, 45]
	5.4	1.1	4.3	1:3.9	HM-1	Neomycin/TK	Undisclosed	[46]

TK: thymidine kinase gene; HAT: hypoxanthine-aminopterin-thymidine; DTA: diphtheria toxin A.

Table 2

Comparison of the sequence features of several widely used loci for site-specific insertion of exogenous gene.

Gene/locus	Length of the 5’ and 3’ homologous arms	G/C content (%) (5’/3’)	SSR (%) (5’/3’)	CpG islands (n) (5’/3’)	Percentage of SINE (%) (5’/3’)	Percentage of LINE (%) (5’/3’)	Other DNA elements (%) (5’/3’)
Myh9	4.0/1.7	50.4/50.8	0.0/0.0	0/0	3.1/0.0	0.0/0.0	0.0/0.0
ColA1	3.3/2.7	51.7/49.7	5.9/2.9	0/0	0.0/0.0	0.0/0.0	0.0/0.0
HPRT	3.8/5.8	43.0/39.3	1.9/7.5	0/0	0.0/0.0	4.5/0.0	0.0/0.0
ROSA26	1.1/4.3	68.3/38.7	0.0/0.0	1*/0	0.0/4.1	0.0/0.0	0.0/2.6

GC content represents the percentage of nucleotides in the strand that possesses either cytosine or guanine bases; Simple sequence repeat (SSR) consists of short, tandemly repeated di, tri-, tetra- or penta-nucleotide motifs; CpG island is a short stretch of DNA in which the frequency of the CG sequences is higher than other regions; SINEs denotes short interspersed nuclear elements; LINEs denotes long interspersed nuclear elements;

*CpG island located at the position of 48–810.

GT efficiency at various targeting sites of the Myh9 gene locus

The effects of loci, including chromosome position and sequence context on gene targeting efficiency are well recognized [17, 37], but the effects of shifting the position of homologous arms on the same locus remain to be explored. Undoubtedly, the property of high GT efficiency at the Myh9 locus is useful for investigating this aspect. For this purpose, a series of constructs targeting to various sites of the Myh9 gene locus including the exon2, intron2 and exon3 positions, were generated. Notably, the constructs targeting to the intron2 and exon3 sites even had slightly longer homologous arms as indicated in Fig 1A since the GT efficiency was suggested to be directly proportional to the length of the homologous arms. Gene targeting experiments with these constructs were performed and HR events were identified as described above (Fig 1B and 1C). In contrast to previous studies [16, 47, 48], our results demonstrated that longer arms are not always associated with higher GC efficiency and also revealed that there is obvious difference in the GT efficiency at various sites even in the same locus. Briefly, the highest GT efficiency occurred at the exon2 site (91.7%), followed by that at the intron2 site (71.8%), while the exon3 site had the lowest efficiency (50.0%), displaying a trend of gradual decrease with the shift of the targeting site from the exon2 to intron2 and exon3 (Table 3). Consistent with a previous idea that the nature of the locus is critical [17], our result further highlighted the importance of the suitable targeting site in the same gene locus for desirable GT frequency. Furthermore, this view was also supported by recent studies, which suggested that even the sites for the application of gene editing tools to some specific loci should be considered carefully [49, 50]. Notably, our previous studies indicated that in spite of high GT frequency at the Myh9 gene locus, both PCR and Southern Blot did not identify ES clones with double knockout alleles [27, 29–31]. The major reason might be that homozygous knockout of the Myh9 gene can cause abnormal proliferation and ESC morphology [51], which in turn result in bias for colony picking, so does the current study.

Fig 1

Gene targeting at various sites of the mouse Myh9 locus.

(A) Diagram of the constructs targeting to different sites of the Myh9 gene locus. Shown are part of the Myh9 gene locus including exons1-4, the distance between them, the down arrows representing the targeted insertion of Neomycin (upper), the constructs targeting to distinct sites of the Myh9 gene locus and the length of the 5’ and 3’ arms of these constructs (middle), as well as the whole sequence region used for the generation of these constructs (lower). The HR efficiency of these targeting constructs is summarized in Table 3. The average percentage of HR efficiency is derived from two repeats. (B) Representative gel image of PCR identification of the GT recombinants at the Myh9 gene exon2 site. (C) Representative gel image of PCR identification of the GT recombinants at the Myh9 gene exon3 site. For representative gel image of PCR identification of the GT recombinants at the Myh9 intron2 site, please see reference [31]. M: DNA Marker; P: Positive control; N: Negative control; PC: positive clone; NC: negative clone.

Table 3

Gene targeting frequency at distinct targeting sites of the Myh9 locus in mouse ES cells.

Targeting site	Positive vs Screened clones	Targeting frequency (%)
exon2	10/12	91.7
	12/12
intron2	11/16	71.8
	12/16
exon3	6/12	50
	6/12

To explore the underlying reasons responsible for obvious difference in the GT efficiency at various sites of the Myh9 locus, the whole sequences used to create these targeting constructs were analyzed in detail as described in Materials and Methods. As summarized in Table 4, the GC-content in these three sequences for constructs targeting to the Myh9 exon2 (50.5%), intron2 (50.6%) and exon3 (51.1%) was only slightly increased with the extended length of the homologous arms, while the percentage of SSR and SINEs in them was markedly decreased. Briefly, the construct targeting to the Myh9 exon2 possessed the highest percentage of SSR (11.1%) and SINEs (9.2%), followed by those of the construct targeting to the intron2 (10.1%, 8.0%, respectively), while the construct targeting to the Myh9 exon3 had the lowest ones (4.2%, 2.3%, respectively). Interestingly, the trends of decreased percentage of SSR and SINEs in the homologous arms of these constructs were completely consistent with that of the GT efficiency for them. Importantly, the potential effects of these elements on gene targeting have been suggested [52-56], though direct evidence remain to be substantiated. Therefore, this finding presented here not only supports the concept, but also provide an excellent locus for in-depth investigating this issue in the future.

Table 4

The bioinformatic analysis of the sequences used to create the constructs targeting to different sites of the Myh9 locus.

The constructs targeting to the sites of the Myh9 locus	The properties of the sequences used to create the targeting construct
	Total length (bp)	GC content (%)	Percentage of simple sequence repeat (SSR) (%)	CpG Island (%)	Percentage of SINEs (%)	Percentage of LINEs (%)	Percentage of other DNA elements (%)
exon2	5630	50.5	11.1	No	9.2	0.0	0.0
intron2	6485	50.6	10.1	No	8.0	0.0	0.0
exon3	6580	51.4	4.2	No	2.3	0.0	0.0

GT efficiency at the Myh9 gene locus with various homologous arm lengths

Earlier investigations suggested that there is a good correlation between GT efficiency and the length of homologous arms [47, 56–59], but another report also showed that increasing the length of the targeting sequences does not always enhance the efficiency of HR [14]. This idea was partially supported by our observation that the construct targeting to the exon2 site of the Myh9 gene locus with a total length of approximate 5.7 kb homologous arms obtained an unprecedented HR frequency [27, 29, 30]. Additionally, our effort to use the constructs with increasing length of homologous arms targeting to distinct sites of the Myh9 gene locus also confirmed this concept, as demonstrated above. Next, two questions related to the targeting events at the Myh9 gene locus were raised about whether this phenomenon is associated with specific sequences in the homologous arms, how long the homologous arms and what the ratio for them are required for optimal GT efficiency. In particular, there is no exact answer for the latter one. On the one hand systematic studies have not been conducted, and on the other hand comparing results generated from different studies is problematic because of the presence of multiple variables in addition to the extent of homology [60]. To answer these questions, a series of truncated targeting constructs with various lengths of the 5’ and 3’ homologous arms were generated as described in Materials and Methods and indicated in Fig 2A. Similarly, PCR was utilized to identify targeting recombinants (Fig 2B). The GT frequency for this series of constructs was summarized in Table 5. Additionally, the trend and relationship of the length of homologous arms and the GT efficiency was analyzed as indicated in Fig 2C. The results indicated: a) With the shortening of the length of the homologous arms either at the 5’ or 3’, the GT frequency displayed a generally downward trend, suggesting the effect of the homologous arm length on the GT efficiency, as consistent with previous reports [57, 59, 60]. Furthermore, the total length of the homologous arms had a positive correlation with the GT frequency (R2 = 0.7256, p<0.01); b) The shortening of 5’ and 3’ arms seemed to have different effects on the GT frequency, overall with that of the former more pronounced than that of the latter; c) Only when the truncated length of the 5’ arm reached one fourth of the original length, the decrease of the targeting efficiency approached an order of magnitude. This differed from a previous report [47] and suggested the effect of the length of the homologous arms on the targeting frequency is complicated and may not be pronounced as anticipated [14]; d) The effect of the absence of the fragment containing the exon2 sequence which is more conserved than others in the 3’ arm on the targeting efficiency was not as large as expected; e) A ratio of 2:1 for the length of the 5’ and 3’ arms seemed better than other combinations (Table 5). Collectively, a targeting construct with a 5.7 kb sequence surrounding the Myh9 gene exon2 as the homologous arms produces a pronounced high GT efficiency in mESCs, while this efficiency is correlated with the length of homologous arms but not to a specific sequence within them. Additionally, an optimal ratio of 2:1 for the length of the homologous arms is suggested even in the presence of gene editing tools for desirable targeting efficiency. Lastly, we examined whether the shortening of 5’ or 3’ homologous arm alters the amount of DNA elements in these sequences, which in turn influences GT efficiency. Similarly, the presence of different DNA elements in each truncated 5’ or 3’ arm was analyzed as above and summarized in Table 6. Firstly, the shortening of 5’ or 3’ arm seemed not to obviously change the GC-content. Secondly, only the shortening of 5’ arm from 4 kb to 2 kb led to a markedly increase of the percentage of SINEs, which in turn alleviates the effect of the shortening of the 5’ arm on GT efficiency. This fact was to some extent consistent with the idea from a previous report [38], which suggests the integration frequency of targeting vector correlates with the amount of SINEs present in the arms.

Fig 2

Gene targeting at the Myh9 gene exon2 site with various length of the homologous arms.

(A) Diagram of a series of truncated targeting constructs. The 5’ homologous arm is left-side truncated from 4, 3, 2 to 1 kb, while the 3’ one is right-side truncated from 1.7, 1.1 to 0.6 kb. Additionally, a left-side truncated 3’ arm with the deletion of the exon2 and beyond (about 0.5 kb) is also generated. Each targeting construct is given a number (No) as indicated at the left, the total length, the length of each 5’ or 3’ arm and the ratio between them are also indicated. Detailed information of these constructs and their HR frequency in mouse ES cells are summarized in Table 5. The average percentage of HR efficiency is derived from two repeats. (B) Shown is representative gel image of PCR identification of the GT recombinants for the targeting construct No4. M: DNA marker; PC: positive clone; NC: negative clone. (C) The graph indicates a downward trend of the gene targeting efficiency with the decreased length of the homologous arms and reflects the correlation of the gene targeting frequency with the total length of the homologous arms, the R2 value shown in the graph is calculated from thirteen points (p<0.01).

Table 5

Summary of the HR frequency for each targeting construct.

Targeting Construct No*	Left arm length (kb)	Right arm length (kb)	Positive vs Total clones	Targeting frequency (%)
0	4.0	1.7	22/24	91.7
1	4.0	1.1	10/24	41.7
2	4.0	0.6	6/24	25.0
3	4.0	1.2	15/24	62.5
4	3.0	1.7	12/24	50.0
5	3.0	1.1	13/24	54.2
6	3.0	0.6	10/23	43.5
7	2.0	1.7	10/24	41.7
8	2.0	1.1	10/24	41.7
9	2.0	0.6	4/24	16.7
10	1.0	1.7	2/24	8.3
11	1.0	1.1	0/24	0.0
12	1.0	0.6	1/24	4.2

Table 6

Genetic features of the sequences used for creating different 5’ and 3’ truncated homologous arms.

Truncated arms (kb)	G+C content (%)	SSR	CpG island	Percentage of SINE (%)	Percentage of LINE (%)	Other DNA elements
5’-4.0	50.4	0.0	No	3.1	0.0	0.0
5’ 3.0	50.2	0.0	No	4.1	0.0	0.0
5’-2.0	51.0	0.0	No	6.1	0.0	0.0
5’-1.0	51.5	0.0	No	0.0	0.0	0.0
3’-1.7	50.9	0.0	No	0.0	0.0	0.0
3’-1.1	50.9	0.0	No	0.0	0.0	0.0
3’-0.6	54.5	0.0	No	0.0	0.0	0.0
3’-1.2	49.2	0.0	No	0.0	0.0	0.0

Conclusion

In the current study we further investigated our previous finding that gene targeting at the Myh9 gene locus has a pronounced high HR frequency. Our study clarified several points and provided some new insight into homologous recombination-based gene targeting. Firstly, through the analysis of the Myh9 and several other widely used loci for targeting transgenesis, the properties of a 6 kb total length, and 50% GC-content present in the homologous arms, may facilitate the achievement of desirable GT efficiency. Secondly, consistent with the central role for the loci, our study also highlights the importance for the selection of an exact targeting site in the practical application of gene targeting, this may be associated with the amount of DNA elements like SINEs in the homologous arms. Thirdly, the targeting efficiency has a positive correlation with the total length of the homologous arms. Moreover, an optimal ratio of 2:1 for the length of the 5’ and 3’ arms with 50% GC-content and the potential effects of DNA elements including SINEs in the arms on GT frequency, are further suggested. Lastly, the in-depth investigations of gene targeting greatly benefit the understanding of the cellular machinery related to HR, the regulation of it, as well as HR-associated factors. The Myh9 gene locus as a potential mitotic recombination hotspot undoubtedly is an ideal molecular and genetical location for further exploration of these aspects.

The PCR primers used in this study.

(DOCX)

Click here for additional data file.

29 Aug 2019

PONE-D-19-15918

Investigation of the molecular biology underlying the pronounced high gene targeting frequency at Myh9 gene locus in mouse embryonic stem cells

PLOS ONE

Dear Dr Wang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Oct 13 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Wenhui Hu, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2.  Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Partly

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: N/A

Reviewer #3: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the manuscript entitled “Investigation of the molecular biology underlying the pronounced high gene targeting frequency at Myh9 gene locus in mouse embryonic stem cells”, the authors perform homologous recombination (HR) in mouse embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and embryonic fibroblasts (MEFs) to investigate the molecular features of the Myh9 gene locus that are responsible for the notably high efficiency of gene targeting. They perform comparative genetic sequence analysis of the Myh9 gene locus to illustrate a low degree of sequence conservation, which in turn suggests there are no unique genetic properties at these loci that affect recombination efficiency. By targeting their vector into the exon 2 region of Myh9 in mouse ESCs, iPSCs, and MEFs, the authors demonstrate a variable HR efficiency that is cell type-dependent but transcriptionally and translationally independent. The efficiency of HR also declines as the targeting vector is shifted downstream of exon 2, highlighting the contributions of local regions to recombination efficiency. Finally, the authors investigate the effects of modifying the lengths of the homology arms and discover an optimal ratio of 5’ to 3’ arm lengths for effective HR. Because gene editing has proven itself as an indispensable tool in biomedical research, primarily in enabling the generation of genetic models of development and disease, it is a worthwhile effort to understand the mechanisms regulating these processes. Despite the enormous strides in gene editing technology over the past decade using programmable nucleases, challenges remain in the field such as increasing the size of the targeting vector or minimizing off target genetic modifications. Understanding the properties of cis and trans factors affecting the efficiency of gene targeting through classical HR can yield mechanistic insights that have positive implications for other gene targeting technologies.

While to goal of this manuscript is to advance the mechanistic understanding of how properties of the Myh9 locus enable such high gene targeting efficiencies through HR, the low experimental robustness and interpretation presented in this manuscript limit the validity of the conclusions drawn. It is by these qualities that this manuscript as submitted is not appropriate for publication in PLOS One, and descriptions of these qualities are outlined below.

First, it is unclear in the written text what the authors aim to convey from the sequence comparisons across different species. Is there prior evidence that sequence conservation predicts strong recombination effects? Is there evidence that HR is high or low in ESCs of other species? If, for instance, HR efficiency is comparable in both rat and mouse ESCs (PMID: 20703227, PMID: 21151976), can conserved sequences be found across both loci? If there are discrepancies in HR efficiency across two species despite conserved sequences within syntenic elements, this could suggest differential epigenetic effectors. The authors can incorporate efficiency data from other ESC studies and better frame the interpretations drawn from their analysis. Furthermore, the data presented in Table 1, specifically the percent sequence similarity among the homology arms should be analyzed separately for the 5’ and 3’ arms. Figure 1A, or any of the other gene diagrams are not to scale and should indicate this.

Next, the authors should report on how many different ESC, iPSC, and MEF clonal lines were used to produce the data presented in Figure 2, as well as what passage number. If a single clonal cell line were used for each, this limits the generalizability of their findings. Furthermore, if these properties change over prolonged passaging of cell lines, this could also explain effects on efficiency. An ideal experiment to truly compare epigenetic states would be to conduct HR experiments on cell types derived from the same genetic individual. For instance, the authors could in vitro differentiate an ESC line into somatic cells such as fibroblasts, then reprogram them into iPSCs, and perform HR experiments on all three epigenetic states. Methodologically, the PCR should be designed in a way to detect a PCR product produced from the wild type alleles, as opposed to seeing no PCR product amplified. When no PCR band is detected for that sample, it could have resulted from a failed PCR run. The agarose gel images should be presented. Additionally, the authors should indicate where and whether the qPCR primers and antibody epitope account for exon 2.

Lastly, what is the contribution of cell cycle of each cell type to the HR efficiency? Could a difference in cell cycling rate between the ESC and iPSC lines examined explain the difference in the rate of successful targeting?

In sum, this manuscript falls short of the technical standards and logical conclusions expected from PLOS publications. Until the authors address the shortcomings described above and write a coherent story based on tenable data, this work should not be published as presented.

Reviewer #2: Tan and co-workers explore the efficiency of traditional gene targeting of the murine Myh9 locus. The work in the manuscript is focused on the mouse Myh9 locus, however, the discussion includes speculation about other species with relatively shallow bioinformatics analysis. Conversely, detailed analysis of the murine Myh9 locus and the sequence of the homology arms lacks any sizable detail. While the identification of the murine Myh9 locus as a mitotic recombination hotspot would be of interest for the field of genome editing, especially for those trying to optimize gene targeting, the manuscript as-is lacks any in depth analysis. The manuscript is both highly speculative about the underlying cause of the high efficiency targeting, while being relatively shallow about the details of the actual molecular work that was done. The manuscript would be greatly improved if it was focused on the mouse Myh9 locus and provided greater details about the various targeting vectors utilized and the genome editing efficiencies observed.

Major Comments

1) The comparison of the Myh9 locus in various species does not provide any insight into the high targeting efficiency of the murine Myh9 locus. In this manuscript, only the mouse genome is targeted, and it is inappropriate to infer anything about the targeting potential of the Myh9 locus in these other species, and the contribution of the flanking genomic sequence and the species-specific Myh9 locus. Unless IPSCs from these various species are also used for homologous gene targeting of the Myh9 locus, there is little information to be gained from comparing the Myh9 loci.

2) Although many aspects of homologous directed targeting remain unknown, it is accepted that critical for homologous targeting is mitosis and proliferation. Therefore, the inability to target the Myh9 locus in MEFs, which have a lower proliferative index and readily senescence in culture, is not surprising. The lack of senescence and high proliferation rate of ESCs and IPSCs is critical for efficient homologous gene targeting. Therefore, the expression level of Myh9 in MEFs is inconsequential in this reviewer’s opinion. The proliferation rate/index of the IPSCs and ESCs used in this study should be compared.

3) With gene targeting efficiency over 50%, it would be expected that some of the clones to be homozygous for the targeted allele. For a locus specific report about homologous targeting, zygosity of targeting should be added to the manuscript.

4) The data presented in Table 4 has a good linear relationship between total homology length and targeting efficiency. As presented in the table, it is difficult to see that relationship.

5) The varying targeting efficiency between the three regions of the murine Myh9 locus discussed in Table 3 warrant some additional sequence analysis, such as GC content, repetitive elements, homology to other location in the murine genome, etc.

6) Was chromatin accessibility assessed for the Myh9 locus? Even if not in the actual ESC line used, it would be interesting to know the status of the chromatin/nucleosome in mESCs of the readily targetable Myh9 locus.

Minor Comments

1) Minor grammatical errors such as line 64: “…is rarely”

2) Line 142/143; the purpose of the sub-cloning is unclear.

3) The choice of using the 129-derived BAC DNA as a template for the homology arms and the use of B6129F1 ES cells for electroporation is unclear given that isogenic homology arms are believed to increase targeting efficiency (Line 68).

4) If available, the targeting efficiency of the Myh9 locus in the absence of negative selection (ganciclovir) would be a useful comparison, since negative selection in practice rarely results in the enrichment of correctly targeted clones that are conceptually expected.

5) Source/method of IPSCs generation not indicated.

Reviewer #3: In this study, the authors intend to investigate the molecular biology underlying for the pronounced high gene targeting frequency at Myh9 gene locus. Numerous factors which affect the HR efficiency were investigated, including chromosome position, transcriptional activity, targeted loci, and the length of the homology arm. And details see the attachment.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Comments to the Author-PONE-D-19-15918.doc

Click here for additional data file.

20 Nov 2019

Response to Reviewers

Reviewer #1: In the manuscript entitled “Investigation of the molecular biology underlying the pronounced high gene targeting frequency at Myh9 gene locus in mouse embryonic stem cells”, the authors perform homologous recombination (HR) in mouse embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and embryonic fibroblasts (MEFs) to investigate the molecular features of the Myh9 gene locus that are responsible for the notably high efficiency of gene targeting. They perform comparative genetic sequence analysis of the Myh9 gene locus to illustrate a low degree of sequence conservation, which in turn suggests there are no unique genetic properties at these loci that affect recombination efficiency. By targeting their vector into the exon 2 region of Myh9 in mouse ESCs, iPSCs, and MEFs, the authors demonstrate a variable HR efficiency that is cell type-dependent but transcriptionally and translationally independent. The efficiency of HR also declines as the targeting vector is shifted downstream of exon 2, highlighting the contributions of local regions to recombination efficiency. Finally, the authors investigate the effects of modifying the lengths of the homology arms and discover an optimal ratio of 5’ to 3’ arm lengths for effective HR. Because gene editing has proven itself as an indispensable tool in biomedical research, primarily in enabling the generation of genetic models of development and disease, it is a worthwhile effort to understand the mechanisms regulating these processes. Despite the enormous strides in gene editing technology over the past decade using programmable nucleases, challenges remain in the field such as increasing the size of the targeting vector or minimizing off target genetic modifications. Understanding the properties of cis and trans factors affecting the efficiency of gene targeting through classical HR can yield mechanistic insights that have positive implications for other gene targeting technologies. While to goal of this manuscript is to advance the mechanistic understanding of how properties of the Myh9 locus enable such high gene targeting efficiencies through HR, the low experimental robustness and interpretation presented in this manuscript limit the validity of the conclusions drawn. It is by these qualities that this manuscript as submitted is not appropriate for publication in PLOS One, and descriptions of these qualities are outlined below.

Answer: All of the authors thank the reviewer for the comments on this manuscript. In the revised version, we perform more detailed bioinformatic analysis, remove the not well addressed part conducted in mESCs, miPSCs and MEFs, and also provide necessary results. Moreover, we answer the concerns/questions raised by the reviewer in a point-to-point way as following.

Question #1: A: First, it is unclear in the written text what the authors aim to convey from the sequence comparisons across different species. B: Is there prior evidence that sequence conservation predicts strong recombination effects? C: Is there evidence that HR is high or low in ESCs of other species? If, for instance, HR efficiency is comparable in both rat and mouse ESCs (PMID: 20703227, PMID: 21151976), can conserved sequences be found across both loci? D: If there are discrepancies in HR efficiency across two species despite conserved sequences within syntenic elements, this could suggest differential epigenetic effectors. The authors can incorporate efficiency data from other ESC studies and better frame the interpretations drawn from their analysis. E: Furthermore, the data presented in Table 1, specifically the percent sequence similarity among the homology arms should be analyzed separately for the 5’ and 3’ arms. Figure 1A, or any of the other gene diagrams are not to scale and should indicate this.

Answer #1: We thank the reviewer for the comments. The questions in this paragraph are split into multiple ones and answered separately.

A: To our knowledge, this is the first time that such a high homologous recombination efficiency was reported and extensively investigated in mammalian cells based on traditional homologous recombination-mediated gene targeting [Liu T et al, PLoS One. 2018]. This property has facilitated the generation of a series of mouse models [Wang A et al, Proc Natl Acad Sci U S A. 2010; Zhang Y et al, Blood. 2012]. This efficiency is even much higher than using much longer homologous arms, e.g bacterial artificial chromosomes (BAC) DNA (6%, Testa G et al. Nat Biotechnol. 2003, 21(4):443-7), or designer nucleases-facilitated (ZFN, TALEN, and CRISPR) gene-targeting (≥20%, Casola S. Methods Mol Biol. 2010, 667:145-63). We have consulted a number of experts who study homologous recombination, but have not gained any useful clues on investigating the underlying mechanisms for this unique phenomenon. HR efficiency is collectively decided by many factors such as the target locus itself, isogenic DNA, length of homologous arms, transcription activity of the target locus, and the status of the ES cells [Ledermann B, Exp Physiol. 2000, 85(6):603-13]. Additionally, previous study indicated that meiotic recombination hotspots in mice are clustered with the major histocompatibility complex (MHC) region (Paul P et al. DNA Repair. 2016, 40:47-56), implying the genetic context matters. Meanwhile, it was suggested that comparative analysis of DNA sequences from multiple species at varying evolutionary distances is a powerful approach for identifying sequences that are unique for a given organism [Frazer KA, et al. Genome Res. 2003;13(1):1-12]. As an initial effort, we compared the genes flanking the Myh9 locus and the Myh9 sequences of mouse used to generate the homology arms with those of other species. The aim of these comparisons is to examine whether the Myh9 gene locus has undescribed and unique properties among different species including the conservation in terms of genetic context and whether there are distinct cis-elements, resembling LoxP, FRT, and other sequences, contributing to this high HR efficiency. The locus and sequence comparison could at least rule out this possibility or provide clues.

B: There is no evidence indicating that sequence conservation predicts higher recombination rate, but there are indications that sequence divergence has an inhibitory effect on homologous recombination [Lukacsovich T et al. Genetics. 1999, 57(1):43-56; Opperman R et al. Genetics. 2004, 168(4):2207-15].

C: Mouse is the only species, in which ES cells have been extensively used in gene-targeting experiments. Germline-competent ES cells have also been generated in rats, but very few gene-targeting experiments have been reported. ES cell lines from some other mammalian species have been reported, but they have not been proven to be able to transmit through the germline, and therefore not used much in gene knockout experiment. No reports or data are available for comparing the targeting efficiency in mouse and rat embryonic stem cells. In general, the ES cells-based gene targeting efficiency is around 1-10% in the species of mouse [Brown AC et al. Cytotechnology. 2006, 51(2):81-8]. As far as the cases mentioned by the reviewer, it seems the HR efficiencies in those two cell types are comparable without considering the strategies and conditions used [Doetschman T et al. Proc Natl Acad Sci U S A. 1988, 85(22):8583-7; Donehower LA et al. Nature. 1992, 356(6366):215-21; Meek S et al. PLoS One. 20105(12):e14225; Tong C et al. Nature. 2010, 467(7312):211-3], in which a targeting efficiency of 1-3.7% can be achieved in the ES cells from both species.

D: It is worth mentioning that most targeting efficiencies reported in the literature are within an expected range, together with multiple determinants for the efficiency, make it difficult to compare these cases. Likewise, a tight and direct relationship between epigenetic factors and homologous recombination(HR)-based gene targeting remains lacking, although there are a lot of studies indicating the contribution of these epigenetic factors to HR-directed DNA repair [Vélez-Cruz R et al. Genes Dev. 2016, 30(22):2500-2512; Lang F et al. Proc Natl Acad Sci U S A. 2017, 114(41):10912-10917; Chakraborty U et al. Genetics. 2019, 212(4):1147-1162]. Undoubtedly, the property of high gene targeting frequency at the MyH9 gene locus reported here facilitates to investigate those interesting questions in the future.

E: Additionally, the bioinformatic analysis of the similarity among the homology arms is further conducted and presented, and the claim of the gene diagrams not to scale is added in the revised version.

Question #2: A: Next, the authors should report on how many different ESC, iPSC, and MEF clonal lines were used to produce the data presented in Figure 2, as well as what passage number. If a single clonal cell line were used for each, this limits the generalizability of their findings. Furthermore, if these properties change over prolonged passaging of cell lines, this could also explain effects on efficiency. B: An ideal experiment to truly compare epigenetic states would be to conduct HR experiments on cell types derived from the same genetic individual. For instance, the authors could in vitro differentiate an ESC line into somatic cells such as fibroblasts, then reprogram them into iPSCs, and perform HR experiments on all three epigenetic states. C: Methodologically, the PCR should be designed in a way to detect a PCR product produced from the wild type alleles, as opposed to seeing no PCR product amplified. When no PCR band is detected for that sample, it could have resulted from a failed PCR run. The agarose gel images should be presented. Additionally, the authors should indicate where and whether the qPCR primers and antibody epitope account for exon2.

Answer #2: We thank the reviewer for the comment. A: Since the similar concern was raised by all reviewers, we remove the part involving the experiment conducted in miPSCs and MEFS after communicating with the Academic editor, Dr Hu. All mouse ESC gene-targeting experiments were conducted using passage 14 (P14) V6.5 ES cell line. Our transgenic core has frozen hundreds of vials of P14 V6.5 ES cells, and we thaw a new vial for each construct. Therefore, all constructs were done using the same passage cells and the cell cycle/proliferation rate should be very similar. B: miPSC and MEF data have been removed. C: The long-range PCR using one primer located at the Neomycin selection maker and one primer resided in the outside of the short arm, has widely been used to identify the gene targeting recombinants in our previous and other studies [Liu T et al, PLoS One. 2018, 13(2):e0192641; Zhou D et al. J Vis Exp. 2018, (141); Lay JM et al. Transgenic Res. 1998, 7(2):135-40; Anastassiadis K et al. Methods Enzymol. 2013533:133-55; Fisher CL et al. Nucleic Acids Res. 2017, 45(21):e174; Sommer D et al. Nat Commun. 2014, 5:3045]. Furthermore, a positive and a negative control were included in each run of PCR to rule out the PCR failure. The PCR products were also sequenced to further confirm the targeting events. Additionally, representative agarose gel images are presented in the revised version, all the PCR primers have been listed in the supplementary table 1.

Question #3: A: Lastly, what is the contribution of cell cycle of each cell type to the HR efficiency? Could a difference in cell cycling rate between the ESC and iPSC lines examined explain the difference in the rate of successful targeting? B: In sum, this manuscript falls short of the technical standards and logical conclusions expected from PLOS publications. Until the authors address the shortcomings described above and write a coherent story based on tenable data, this work should not be published as presented.

Answer #3: We thank the reviewer for the comment. A: As stated above, we have removed the experiments conducted in miPSCs and MEFs per the permission of the academic editor, Dr. Hu, though our observation of the gene targeting efficiency in mouse ES cells higher than that in iPS cells is to some degree consistent with a previous study [Zou J et al. Cell Stem Cell. 2009, 5(1):97-110]. B: We believe that our revised version has been improved and should reach the standard of the journal and the response to the comments facilitates to understand the story of this manuscript.

Reviewer #2: Tan and co-workers explore the efficiency of traditional gene targeting of the murine Myh9 locus. The work in the manuscript is focused on the mouse Myh9 locus, however, the discussion includes speculation about other species with relatively shallow bioinformatics analysis. Conversely, detailed analysis of the murine Myh9 locus and the sequence of the homology arms lacks any sizable detail. While the identification of the murine Myh9 locus as a mitotic recombination hotspot would be of interest for the field of genome editing, especially for those trying to optimize gene targeting, the manuscript as is lacks any in depth analysis. The manuscript is both highly speculative about the underlying cause of the high efficiency targeting, while being relatively shallow about the details of the actual molecular work that was done. The manuscript would be greatly improved if it was focused on the mouse Myh9 locus and provided greater details about the various targeting vectors utilized and the genome editing efficiencies observed.

Answer: All of the authors thank the reviewer for the comments on this manuscript. In the revised version, we perform more detailed bioinformatic analysis, remove the not well addressed part conducted in mESCs, miPSCs and MEFs, and also provide necessary results. Moreover, we answer the concerns/questions raised by the reviewer in a point-to-point way as following.

Major Comments

Question #1): The comparison of the Myh9 locus in various species does not provide any insight into the high targeting efficiency of the murine Myh9 locus. In this manuscript, only the mouse genome is targeted, and it is inappropriate to infer anything about the targeting potential of the Myh9 locus in these other species, and the contribution of the flanking genomic sequence and the species-specific Myh9 locus. Unless IPSCs from these various species are also used for homologous gene targeting of the Myh9 locus, there is little information to be gained from comparing the Myh9 loci.

Answer #1: The authors thank the reviewer for this suggestion. 1) it was suggested that comparative analysis of DNA sequences from multiple species at varying evolutionary distances is a powerful approach for identifying sequences that are unique for a given organism [Frazer KA, et al. Genome Res. 2003;13(1):1-12]. 2) The aim of these comparisons is to examine whether the Myh9 gene locus has undescribed and unique properties among different species including the conservation in terms of genetic context and whether there exist unique elements like LoxP element contributing to this high HR efficiency. The locus and sequence comparison could at least rule out this possibility or provide other clues. 3) To our knowledge, it is the first time to report a so high homologous recombination efficiency at the Myh9 gene locus in mouse embryonic stem cells based on traditional gene targeting [Liu T et al, PLoS One. 2018], this property has facilitated the generation of a series of mouse models [Wang A et al, Proc Natl Acad Sci U S A. 2010; Zhang Y et al, Blood. 2012]. This efficiency is also much higher than that of even using longer homology arms, e.g bacterial artificial chromosomes (BAC) DNA (6%, Testa G et al. Nat Biotechnol. 2003, 21(4):443-7) and that of widely used safe harbor site ROSA26 in mouse embryonic stem cells (≥20%, Casola S. Methods Mol Biol. 2010, 667:145-63). We have even consulted with several experts in the field. Frankly to say, we could not get useful clues to investigate the underlying mechanisms for this unique phenomenon. HR efficiency is collectively decided by many factors such as the target locus itself, isogenic DNA, length of homologous arms, transcription activity of the target locus, and the status of the ES cells [Ledermann B, Exp Physiol. 2000, 85(6):603-13]. Additionally, previous study indicated that meiotic recombination hotspots in mice are clustered with the major histocompatibility complex (MHC) region (Paul P et al. DNA Repair. 2016, 40:47-56), implying the genetic context matters. As the initial effort, we compared the genes flanking the Myh9 locus and the mouse Myh9 sequences used to generate the homology arms with those of other species. 4) The detailed comparisons revealed that the mouse Myh9 gene locus is not unique in terms of genetic context including its flanking genes and gene length, while the sequences used to create the homology arms have higher similarity with that of Rat than with those of other species, moreover, the homology of the right arm sequence is higher than that of the left one. Additionally, the right arm is GC rich relative to the left arm and those from other species. Further investigations are warranted to determine whether these properties are associated with the GT efficiency.

Question #2): Although many aspects of homologous directed targeting remain unknown, it is accepted that critical for homologous targeting is mitosis and proliferation. Therefore, the inability to target the Myh9 locus in MEFs, which have a lower proliferative index and readily senescence in culture, is not surprising. The lack of senescence and high proliferation rate of ESCs and IPSCs is critical for efficient homologous gene targeting. Therefore, the expression level of Myh9 in MEFs is inconsequential in this reviewer’s opinion. The proliferation rate/index of the IPSCs and ESCs used in this study should be compared.

Answer #2: The authors thank the reviewer for this concern. Indeed, the cell cycle is an important factor affecting GT efficiency [Majumdar A et al. J Biol Chem. 2003, 278(13):11072-7]. Since the point could not well addressed in the study, we remove the part involving the experiment conducted in miPSCs and MEFS after communicating with the Academic editor, Dr Hu.

Question #3): With gene targeting efficiency over 50%, it would be expected that some of the clones to be homozygous for the targeted allele. For a locus specific report about homologous targeting, zygosity of targeting should be added to the manuscript.

Answer #3: The V6.5 ESCs were derived from F1 hybrid mice, with 50% 129 and 50% C57BL/6, while the targeting constructs were made using 129 genomic DNA. Therefore, the constructs are isogenic to one of the two chromosomes. Importantly, targeting only one chromosome is advantageous in this case because homozygous knockout of Myh9 can cause abnormal proliferation and ESC morphology [Conti MA et al. J Biol Chem. 2004;279(40):41263-6], which can result in bias for colony picking. No ES clones with double knockout alleles were identified whether using PCR or Southern Blot methods [Wang A et al. Proc Natl Acad Sci U S A. 2010;107(33):14645-50; Zhang Y et al. Blood. 2012;119(1):238-50; Liu T et al. PLoS One. 2018;13(2):e0192641]. This information has been added in the revised version.

Question #4): The data presented in Table 4 has a good linear relationship between total homology length and targeting efficiency. As presented in the table, it is difficult to see that relationship.

Answer #4: Thanks for the useful suggestion. To better indicate the relationship between total length of homology arms and GT efficiency, we have reorganized the data and provided more detailed analysis. Indeed, there exists a linear relationship between those two, as shown in Figure 3 in the revised version.

Question #5): The varying targeting efficiency between the three regions of the murine Myh9 locus discussed in Table 3 warrant some additional sequence analysis, such as GC content, repetitive elements, homology to other location in the murine genome, etc.

Answer #5: Thank the reviewer for this good suggestion. We performed further sequence analysis of the constructs targeting to different sites of the Myh9 gene locus, including GC content, CpG island, simple sequence repeat (SSR), SINE, LINE, or other DNA elements, as summarized in the table 3 in the revised version. Interestingly, there is a consistency between the percentage of SSR and SINE and the efficiency of GT. No significant similarity between the sequences used to create these targeting constructs and the sequences of other regions in the mouse genome was observed.

Question #6): Was chromatin accessibility assessed for the Myh9 locus? Even if not in the actual ESC line used, it would be interesting to know the status of the chromatin/nucleosome in mESCs of the readily targetable Myh9 locus.

Answer #6: Thank the reviewer for the comment. Frankly to say, the information about the status of the chromatin accessibility at the Myh9 locus in mESCs could not facilitate to explain the readily targetable Myh9 locus. On one hand, it has been suggested that the chromatin state influences the targeting or editing efficiency mediated by Triplex-forming oligonucleotides (TFOs) or genome editing tools [Macris MA et al. J Biol Chem. 2003 Jan 31;278(5):3357-62; van Rensburg R et al. Gene Ther. 2013 Feb;20(2):201-14; Daboussi F et al. Nucleic Acids Res. 2012 Jul;40(13):6367-79], while no evidence indicate the effect of chromatin accessibility on traditional HR-based gene targeting. On the other hand, earlier studies demonstrated that GT frequency is independent of target gene transcription [Johnson RS et al. Science. 1989,245(4923):1234-6; Yáñez RJ et al. Gene Ther. 1998, 5(2):149-59]. More importantly, our previous studies indicated that the expression level of the Myh9 gene in mESCs is considerable [Conti MA et al. J Biol Chem. 2004,279(40):41263-6; Wang A et al. Proc Natl Acad Sci U S A. 2010;107(33):14645-50; Zhang Y et al. Blood. 2012;119(1):238-50; Liu T et al. PLoS One. 2018;13(2):e0192641].

Minor Comments

1) Minor grammatical errors such as line 64: “…is rarely”

A: This error has been corrected. Furthermore, we have also corrected some other grammatical errors during revision.

2) Line 142/143; the purpose of the sub-cloning is unclear.

A: The purpose of cloning PCR products into T-easy for sequencing is to make sure the GT recombinants identified by PCR are absolutely right ones. Because one primer is located at the 3’end of Neomycin and another one resides outside of the short arm, the sequencing result can confirm this.

3) The choice of using the 129-derived BAC DNA as a template for the homology arms and the use of B6129F1 ES cells for electroporation is unclear given that isogenic homology arms are believed to increase targeting efficiency (Line 68).

A: The V6.5 ESCs were derived from F1 hybrid mice, with 50% 129 and 50% C57BL/6, while the targeting constructs were made using 129 genomic DNA. Therefore, the constructs are isogenic to one of the two chromosomes. Importantly, targeting only one chromosome is advantageous in this case because homozygous knockout of Myh9 can cause abnormal proliferation and ESC morphology [Conti MA et al. J Biol Chem. 2004;279(40):41263-6], which can result in bias for colony picking.

4) If available, the targeting efficiency of the Myh9 locus in the absence of negative selection (ganciclovir) would be a useful comparison, since negative selection in practice rarely results in the enrichment of correctly targeted clones that are conceptually expected.

A: We had no data for GT efficiency at the Myh9 gene locus in the absence of ganciclovir.

5) Source/method of IPSCs generation not indicated.

A: After communicating with the academic editor Dr. Hu, we decided to remove the part involving the experiments conducted in miPSCs and MEFs, thus, the source/method of iPSCs generation is no longer needed.

Reviewer #3: In this study, the authors intend to investigate the molecular biology underlying for the pronounced high gene targeting frequency at Myh9 gene locus. Numerous factors which affect the HR efficiency were investigated, including chromosome position, transcriptional activity, targeted loci, and the length of the homology arm. Some issues should be addressed or discussed in detail below.

A: The authors thank the reviewer for providing comments on this manuscript. The concerns or issues are addressed below and in the revised version.

Major comments:

1. Authors constructed 5’ and 3’ homologous arm from the 129/Sv genomic BAC clone DNA, and then the mouse cells from hybrid C57BL/6 and 129S6/SvEv were targeted. For sequence divergence among laboratory mice and the divergence imposed a major handicap in mutating the gene through homologous recombination (see HEIN TE RIELE, 1992, ref#15), it is possible that the cells line background affects the HR efficiency. The authors should sequence the target locus of cell lines to ensure that is consistent with the homologous arm sequences.

Answer #1: Thanks for this comment. The V6.5 ESCs were derived from F1 hybrid mice, with 50% 129 and 50% C57BL/6, while the targeting constructs were made using 129 genomic DNA. Therefore, the constructs are isogenic to one of the two chromosomes. Importantly, targeting only one chromosome is advantageous at least in this case because homozygous knockout of Myh9 can cause abnormal proliferation and ESC morphology [Conti MA et al. J Biol Chem. 2004;279(40):41263-6], which can result in bias for colony picking. Additionally, no ES clones with double alleles knocked out were identified whether using PCR or Southern Blot method.

2. Authors declared that “No animal experiments were conducted, no ethic permits were therefore needed for this report”, however, “MEFs were isolated and cultured embryonic day 9.5 to 10.5 (line 130, ref#29)”.

Answer #2: Thanks for reminding this point. We are sorry for not paying attention to it. After communicating with the academic editor Dr Hu, we decide to remove the part involving the experiments conducted miPSCs and MEFs, thus, the source/method of MEFs generation should be removed too. Thus, no animal experiments are indeed involved.

3. The authors did analyze the locus of the Myh9 gene in multiple species. However, there is no data showing the GT frequency in these species. Therefore, it’s hardly to reach the conclusion that low correlation with the high GT frequency at the Myh9 gene in term of chromosome position from the variable or conserved the locus Myh9 and its flanking genes. And several other types of genomic elements should be analyzed which may affect GT efficiency in Myh9, including CpG islands, simple repeats, microsatellites, DNA transposons, SINEs, LINEs et al.

Answer #3: Thanks for this good suggestion. According to the suggestion, we have performed further bioinformatic analysis on the sequences used to create construct targeting to the Myh9 gene exon2 in the mouse genome and the corresponding sequences from other species, the results are summarized and presented in the revised version.

4. The authors declared that the length and ratio of homologous arms affected the GT efficiency (Table 4). However, when targeting the Myh9 gene Exon 2, Intron 2 and Exon 3, why choose similar length of the homologous arms rather than the same length? And Dose the difference length and ratio of homologous arms also lead to the results?

Answer #4: Thanks for this inquiry. Firstly, a 11.5kb region spanning from the upstream of exon2 to the downstream of the exon3 was used to create those three targeting constructs. These constructs were made at different times. Due to the availability of restriction sites and PCR primer sequences, sometimes it is hard to make constructs with exact the same length. The GT efficiency is determined by multiple factors, such as the locus, the length of the homologous arms [Ledermann B, Exp Physiol. 2000, 85(6):603-13]. Thirdly, according to previous studies and our observation, the length and ratio of the homologous arms influences the GT efficiency. In current case, the length and ratio of the homologous arms of the constructs targeting to the Myh9 exon2, intron2 and exon3 was slightly increased, which were done unintentionally. Surprisingly, the GT efficiency is contrary to the length and ratio of the homologous arms (Figure 2 and table 2), suggesting other factors are responsible for this.

Minor comments:

5. In Figure 1A, The Unch is short for? In Figure 1B shows the Divergence but the Figure legend is similarity.

Answer #5: Thanks for the inquiry and correction. The Unch is short for uncharacterized, as indicated in the figure legend. In Figure1B, both the percentage of divergence and similarity are indicated.

6. In Figure 2A, what does the black triangle indicates? In Figure 2B and 2D, the mRNA level and the protein level of NMHC IIA in ES cells were set as 100%, the SD should not exist in ES cells columns.

Answer #6: Thanks for the inquiry. The part involving the experiments conducted in mESCs, miPSCs and MEFs has been removed from the manuscript after communicating with the academic editor Dr Hu.

7. Authors should provide the data of identify recombination events

Answer #7: Thanks for the suggestion. Representative images of PCR identification of HR events were provided in the revised version.

8. The antibodies company should be labeled.

Answer #8: Thanks for the suggestion, but it is unnecessary since that part has been removed.

9. Many grammatical or spelling should be corrected thoroughly in the manuscript.

Answer #9: Thanks for this suggestion. The manuscript has been revised and the grammatical or spelling errors has been checked by a native speaker Dr Adelstein (NIH/NHLBI).

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

10 Dec 2019

PONE-D-19-15918R1

Investigation of the molecular biology underlying the pronounced high gene targeting frequency at the Myh9 gene locus in mouse embryonic stem cells

PLOS ONE

Dear Dr Wang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by January 10, 2020. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Wenhui Hu, M.D., Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Tan et al. have removed and analyzed existing data to address reviewers’ concerns about the original draft of their manuscript. Their existing findings and conclusions relating gene targeting (GT) efficiency to cell type (i.e. ESC, iPSC, and MEF) have been removed to focus on the mechanisms underlying GT at the mouse Myh9 locus in ESCs. In response to reviewers’ suggestions, the authors have also analyzed genome sequence elements such as CG content, SSR, and SINE to consider functional elements possibly contributing to the high GT efficiency, specifically at the immediate regions flanking exon 2, which serve as the homology arms. They present this analysis on other species for comparison as well as present sequence elements for their other targeting sequences at intron 2 and exon 3. They have also presented gel images in the figures to show the outcome of their PCR results. Finally, the authors performed correlation analysis between total length of homology arms and targeting efficiency to quantitatively demonstrate a relationship.

The major concern remaining with the currently revised manuscript is the relevance of the genomics sequence comparison with other species. All three reviewers raised this concern and recognized that without GT efficiency data from other species, the comparison of genomic features is meaningless. The cross-species comparison of the locus sequence similarity is not an effective method to convey the mechanism of the high GT efficiency. One cannot draw any association between genetic features to function when data on function is completely not presented. This effort is better applied to compare genomics features among the Myh9 locus with other mouse loci that have regularly been used for GT in mouse ESCs, such as Rosa26, HPRT, or Col1a1, where the GT efficiency has been reported by other groups (PMID: 25143803, PMID: 17183668, PMID: 16400644, PMID: 19141541). While it would be more scientifically rigorous to for the authors to perform these GT experiments at these other loci themselves in the same cell lines they have reported on in this manuscript, they can perform meta-analysis by drawing associations based on data previously published by other labs based on their targeting constructs.

Despite this reviewer’s opinion that the species comparisons should not be performed and that the data do not support the authors’ conclusion, there are critical misinterpretations of the data as they are currently presented:

The methods section describes analyzing only the flanking 5’ and 3’ sequences of Myh9 exon 2 in all species. This is not presented in Figure 1A, which makes it rather misleading to think that Figures 1B, C, and D are based on Figure 1A. In Figure 1A, why is the orientation of the Myh9 loci reversed for the Pig and the Rabbit? This is confusing when the authors refer the right and left arm, rather than the 5’ and 3’ sequences. In Figure 1B, C, and D, the relationships are simply reflecting what is expected to be the relationship on a genome wide level, therefore one should not be surprised that mouse and rat bear such a high degree of similarity. Also, the algorithm for calculating percent identity and divergence should be clearly described for the reader.

Figure 2: PCR data is missing for intron 2.

Figure 3 is mislabeld as Figure 4.

The targeting construct series can benefit the reader by listing properties of each construct to the left of each diagram, such as combined length of homology arms, the ratio of 5’:3’, etc. the length of each arm should also be displayed next to each line.

Figure 3C is basically Figure 3D, but it is not appropriate to apply this type of graphing to this data set. The authors should only present scatter plots in the style of Figure 3D.

In Figure 3D, the authors perform a quantitative measure of the relationship between total length of homology arms and GT efficiency. They can test the significance of this correlation.

The authors should also perform similar quantifiable analysis using the ratio of 5’:3’ lengths, GC content, SSR, and SINE of total, and individual homology arms. These analyses may provide some more insight into the mechanisms underlying the respective GT efficiencies.

Table 4. Why is only one repeat shown? Or is this the sum of 2 repeats? Authors should specify.

Altogether, the manuscript as presented lack sufficient data to model a hypothesis, much less support a detailed, mechanistic understanding of why the Myh9 locus at exon 2 is such an efficient site for GT. The suggested, more in-depth analyses could bring this manuscript to a body of work suitable for publication in PLOS One.

Reviewer #2: The manuscript is greatly improved, and is much more focused on the underlying data and findings. The level of detail provided will allow others to potentially use the murine Myh9 locus as a site to further optimize gene targeting. My only remaining concern (which was shared by other initial reviewers) is the comparison of the murine Myh9 locus with that of other species. With zero gene targeting information for the Myh9 locus in these other species, this is just a distraction for the reader. The inbred mouse is an invaluable tool in biomedical research, and this manuscript should focus on the murine Myh9 locus without speculating about orthologous sequence and how that relates or does NOT relate to the gene targeting. Furthermore, it is misleading to assume that "No unique features and low sequence similarity are found in the Myh9 gene" when comparing to other Myh9 loci for which there is zero gene targeting information. Perhaps a better comparison would be to randomly sample other murine paralogs or similar length/structure loci to see if there is something unique to the murine Myh9 locus that affords such high gene targeting efficiency.

Reviewer #3: Tan et al have partially addressed some concerns issued on their manuscript, "Investigation of the molecular biology underlying the pronounced high gene targeting frequency at the Myh9 gene locus in mouse embryonic stem cells." The reasons for similar length of the homologous arms have been properly elucidated. And the further bioinformatic analysis have been performed. However, some issues still should be addressed or discussed to make the manuscript fit for publication in plos one.

Comment-1

As mentioned before, the authors analyzed the locus of the Myh9 gene in multiple species. While there is no data showing the GT frequency in these species. Therefore, it’s hardly to reach the conclusion that low correlation with the high GT frequency at the Myh9 gene in term of chromosome position from the variable or conserved the locus Myh9 and its flanking genes.

Comment-2

The authors used the V6.5 ESCs which were derived from F1 hybrid mice, with 50% 129S6/SvEv and 50% C57BL/6 for gene targeting, and declared the constructs are isogenic to one of the two chromosomes, while there is no data shows the homology between 129S6/SvEv and C57BL/6 in this loci. Is there evidence that HR is occurs only in the Myh9 loci of 129S6/SvEv? If not, maybe there were some homozygous clones for the targeted allele, which may result in bias for colony picking, as the authors mention, for homozygous knockout of Myh9 can cause abnormal proliferation and ESC morphology. If homology is low, is the targeting efficiency of the Myh9 loci in C57BL/6 as high as 129S6/SvEv?

Comment-3

Unformal writing of gene name such as line 40 “…Myh9 (Italic), line 170 “…MYH9” and Figure 1A. In Figure 3C, the units of vertical and horizontal axis should be provided. In Figure 3D, as targeting efficiency will not be lower than 0, it is more appropriate to set the minimum value of the vertical axis to 0.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

23 Jan 2020

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Tan et al. have removed and analyzed existing data to address reviewers’ concerns about the original draft of their manuscript. Their existing findings and conclusions relating gene targeting (GT) efficiency to cell type (i.e. ESC, iPSC, and MEF) have been removed to focus on the mechanisms underlying GT at the mouse Myh9 locus in ESCs. In response to reviewers’ suggestions, the authors have also analyzed genome sequence elements such as CG content, SSR, and SINE to consider functional elements possibly contributing to the high GT efficiency, specifically at the immediate regions flanking exon 2, which serve as the homology arms. They present this analysis on other species for comparison as well as present sequence elements for their other targeting sequences at intron 2 and exon 3. They have also presented gel images in the figures to show the outcome of their PCR results. Finally, the authors performed correlation analysis between total length of homology arms and targeting efficiency to quantitatively demonstrate a relationship.

Answer: All the authors thank the reviewer for appreciating the improvement of the manuscript.

The major concern remaining with the currently revised manuscript is the relevance of the genomics sequence comparison with other species. All three reviewers raised this concern and recognized that without GT efficiency data from other species, the comparison of genomic features is meaningless. The cross-species comparison of the locus sequence similarity is not an effective method to convey the mechanism of the high GT efficiency. One cannot draw any association between genetic features to function when data on function is completely not presented. This effort is better applied to compare genomics features among the Myh9 locus with other mouse loci that have regularly been used for GT in mouse ESCs, such as Rosa26, HPRT, or Col1a1, where the GT efficiency has been reported by other groups (PMID: 25143803, PMID: 17183668, PMID: 16400644, PMID: 19141541). While it would be more scientifically rigorous to for the authors to perform these GT experiments at these other loci themselves in the same cell lines they have reported on in this manuscript, they can perform meta-analysis by drawing associations based on data previously published by other labs based on their targeting constructs.

Answer: Thanks to the reviewer for this good suggestion! After serious consideration, we decide to remove the comparison of the Myh9 gene locus and the sequences used to create the construct targeting the Myh9 locus with the counterparts from other species. On the one hand, it more seems to be negative data; a functional confirmation of these genetic feature is lacking, on the other hand. According to the suggestion from this reviewer, in the new version, we collected related information about gene targeting at the Rosa26, HRPT, ColA1 loci suggested by the reviewer. The summarization and comparison of gene targeting events and genetic sequences at these loci with those at the Myh9 locus. These contents were described and presented in the new version. Though no common genetic characteristics could be identified among those four loci, a typical 6kb total length for the homologous arms, a 2:1 ratio for the length of the 5’ to 3’ homologous arm, similar and 50% GC-content for the 5’ and 3’ arms, seemed to have better performance, as further confirmed in our subsequent experiments.

Despite this reviewer’s opinion that the species comparisons should not be performed and that the data do not support the authors’ conclusion, there are critical misinterpretations of the data as they are currently presented:

Answer: Thanks a lot. Since the comparison and related data were removed, there was no this issue in the revised version.

The methods section describes analyzing only the flanking 5’ and 3’ sequences of Myh9 exon 2 in all species. This is not presented in Figure 1A, which makes it rather misleading to think that Figures 1B, C, and D are based on Figure 1A. In Figure 1A, why is the orientation of the Myh9 loci reversed for the Pig and the Rabbit? This is confusing when the authors refer the right and left arm, rather than the 5’ and 3’ sequences. In Figure 1B, C, and D, the relationships are simply reflecting what is expected to be the relationship on a genome wide level, therefore one should not be surprised that mouse and rat bear such a high degree of similarity. Also, the algorithm for calculating percent identity and divergence should be clearly described for the reader.

Answer: Thanks to the reviewer for this concern. Similarly, the part related to the comparison and analysis of the Myh9 locus and sequences with those of other species has been removed, the concern from the reviewer was not an issue.

Figure 2: PCR data is missing for intron 2.

Answer: Because a representative image for PCR identification of targeting events at the Myh9 intron2 site has been presented in our previous report [Liu T et al. PLos One, 2018], similar result was therefore omitted in this study.

Figure 3 is mislabled as Figure 4.

Answer: Thanks for the correction. All the figures have been reorganized and marked clearly.

The targeting construct series can benefit the reader by listing properties of each construct to the left of each diagram, such as combined length of homology arms, the ratio of 5’:3’, etc. the length of each arm should also be displayed next to each line.

Answer: Thanks for the good suggestion. Related information has been added and this figure has been modified.

Figure 3C is basically Figure 3D, but it is not appropriate to apply this type of graphing to this data set. The authors should only present scatter plots in the style of Figure 3D.

Answer: Thanks for the good suggestion. This graph has been modified in the new version.

In Figure 3D, the authors perform a quantitative measure of the relationship between total length of homology arms and GT efficiency. They can test the significance of this correlation.

Answer: Thanks for the suggestion. The test of the correlation significance (Represented by p<0.01) between the total length of homologous arms and the GT efficiency has been performed and presented in the revised version.

The authors should also perform similar quantifiable analysis using the ratio of 5’:3’ lengths, GC content, SSR, and SINE of total, and individual homology arms. These analyses may provide some more insight into the mechanisms underlying the respective GT efficiencies.

Answer: Thanks a lot for this good suggestion. The genetic feature analysis of the sequences used for creating different length homologous arms has been conducted, which is summarized and presented in the new version.

Table 4. Why is only one repeat shown? Or is this the sum of 2 repeats? Authors should specify.

Answer: Thanks for raising this concern. This point has been specified in the figure legends of the new version.

Altogether, the manuscript as presented lack sufficient data to model a hypothesis, much less support a detailed, mechanistic understanding of why the Myh9 locus at exon 2 is such an efficient site for GT. The suggested, more in-depth analyses could bring this manuscript to a body of work suitable for publication in PLOS One.

Answer: Thanks a lot for the comments. According to the comments and suggestions from this reviewer, the contents have been adjusted, the related information has been provided, and the figures have been modified, therefore, the manuscript should be greatly improved and reach to the requirement for the journal of PLoS One.

Reviewer #2: The manuscript is greatly improved, and is much more focused on the underlying data and findings. The level of detail provided will allow others to potentially use the murine Myh9 locus as a site to further optimize gene targeting. My only remaining concern (which was shared by other initial reviewers) is the comparison of the murine Myh9 locus with that of other species. With zero gene targeting information for the Myh9 locus in these other species, this is just a distraction for the reader. The inbred mouse is an invaluable tool in biomedical research, and this manuscript should focus on the murine Myh9 locus without speculating about orthologous sequence and how that relates or does NOT relate to the gene targeting. Furthermore, it is misleading to assume that "No unique features and low sequence similarity are found in the Myh9 gene" when comparing to other Myh9 loci for which there is zero gene targeting information. Perhaps a better comparison would be to randomly sample other murine paralogs or similar length/structure loci to see if there is something unique to the murine Myh9 locus that affords such high gene targeting efficiency.

Answer: Thanks to the reviewer for appreciating the improvement of this manuscript and providing other useful suggestions. We have removed the comparison of the Myh9 locus/Sequences from multiple species. Instead of these, we collected, organized and compared the targeting efficiency and the genetic features of several loci often used for site-specific integration, as also suggested by other reviewers. The new content is provided in the revised version.

Reviewer #3: Tan et al have partially addressed some concerns issued on their manuscript, "Investigation of the molecular biology underlying the pronounced high gene targeting frequency at the Myh9 gene locus in mouse embryonic stem cells." The reasons for similar length of the homologous arms have been properly elucidated. And the further bioinformatic analysis have been performed. However, some issues still should be addressed or discussed to make the manuscript fit for publication in plos one.

Answer: Thanks to the reviewer for appreciating the improvement of this manuscript and providing other useful comments.

Comment-1

As mentioned before, the authors analyzed the locus of the Myh9 gene in multiple species. While there is no data showing the GT frequency in these species. Therefore, it’s hardly to reach the conclusion that low correlation with the high GT frequency at the Myh9 gene in term of chromosome position from the variable or conserved the locus Myh9 and its flanking genes.

Answer: Thanks for raising this concern. We have deleted the comparison of the Myh9 locus/Sequences from multiple species. Instead of these, we collected, organized and compared the targeting efficiency and the genetic features of several loci often used for site-specific integration, as also suggested by other reviewers. The new content is provided in the revised version.

Comment-2

The authors used the V6.5 ESCs which were derived from F1 hybrid mice, with 50% 129S6/SvEv and 50% C57BL/6 for gene targeting, and declared the constructs are isogenic to one of the two chromosomes, while there is no data shows the homology between 129/Sv and C57BL/6 in this loci. Is there evidence that HR is occurs only in the Myh9 loci of 1296/Sv? If not, maybe there were some homozygous clones for the targeted allele, which may result in bias for colony picking, as the authors mention, for homozygous knockout of Myh9 can cause abnormal proliferation and ESC morphology. If homology is low, is the targeting efficiency of the Myh9 loci in C57BL/6 as high as 129S/Sv?

Answer: Thanks for raising this concern. The BAC DNA used as the template for obtaining the homologous arms is derived from a 129/Sv genetic background. Importantly, the blast of the whole sequences used for creating the homologous arms indicated that the sequences are100% matching with these from a C57BL/6 genetic background (using the NCBI/Blast program). In other words, the sequences used for creating the targeting constructs are isogenic to both chromosomes. Likewise, no homozygous ES clones were identified by either PCR or Southern Blot.

Comment-3

Unformal writing of gene name such as line 40 “…Myh9 (Italic), line 170 “…MYH9” and Figure 1A. In Figure 3C, the units of vertical and horizontal axis should be provided. In Figure 3D, as targeting efficiency will not be lower than 0, it is more appropriate to set the minimum value of the vertical axis to 0.

Answer: Thanks for these corrections. We have corrected all of them.

Submitted filename: Rebuttal letter.docx

Click here for additional data file.

17 Feb 2020

PONE-D-19-15918R2

Investigation of the molecular biology underlying the pronounced high gene targeting frequency at the Myh9 gene locus in mouse embryonic stem cells

PLOS ONE

Dear Dr Wang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the minor points raised during the second review process.

We would appreciate receiving your revised manuscript by March 1, 2020. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Wenhui Hu, M.D., Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The manuscript in its second revised form is greatly improved. The removal of speculative gene targeting of the Myh9 locus in other species from the main manuscript places focus on the actual work done. Supplementary Figure 1 is poor resolution (and completely unnecessary). While I understand that the multi-species Myh9 locus alignment was time consuming to generate, it really does not add anything of value to the manuscript.

Figure 2C and 2D are the exact same data. there is no need to present the same data twice, just add a trend line to 2C (scale on 2D is nonsensical, -20% targeting efficiency?)

throughout the paper the Myh9 introns and exons are capitalized and written both with and without a space before the number (e.g. Exon2, Exon 2, Intron2, Intron2, etc). Intent is obvious, but it should be at least consistent within the manuscript. Decision to capitalize intron and exon is questionable, but not a scientific issue.

Reviewer #3: Tan et al. have removed the comparison of the Myh9 locus from multiple species, and according to reviewers’ suggestion, the authors compared the length of homologous arms and genomics elements (CG content, SSR, CpG islands, Line, SINE) of the Myh9 locus with other mouse loci (Rosa26, HPRT, and Col1a1) that have widely been used for GT to reveal characteristics potentially contributing to the high GT efficiency. They have also analyzed genomics features of the truncated homologous arms.

Comment-1

The author removed the comparison of the Myh9 locus from multiple species, however, retains the alignment of these sequences in Supplementary Figure 1, and it may more preferably to removed Supplementary Figure 1 together.

Comment-2

Without GT efficiency data from other mouse loci with different ration of homologous arms, it is inappropriate to infer 2:1 ratio for the length of the 5’ and 3’ homologous arms facilitate GT efficiency from the result of comparison the Myh9 locus with other loci.

Comment-3

As other reviewer’ suggestion, Figure 3C is basically Figure 3D, the authors should only present Figure 3D and removed Figure 3C. The concern remaining in the Figure 3D, as targeting efficiency will not be lower than 0, it is more appropriate to set the minimum value of the vertical axis to 0.

Comment-4

In Figure 2A, No.2 total length is 4.6 kb, and the right homologous arm of No.3 should be left aligned.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

21 Feb 2020

Response to reviewers

Reviewer #1: (No Response)

-Reviewer #2: The manuscript in its second revised form is greatly improved. The removal of speculative gene targeting of the Myh9 locus in other species from the main manuscript places focus on the actual work done. Supplementary Figure 1 is poor resolution (and completely unnecessary). While I understand that the multi-species Myh9 locus alignment was time consuming to generate, it really does not add anything of value to the manuscript.

Answer：All the authors thank the reviewer for appreciating the improvement of the revised manuscript. The supplementary Figure 1 has been removed from this manuscript.

-Figure 2C and 2D are the exact same data. there is no need to present the same data twice, just add a trend line to 2C (scale on 2D is nonsensical, -20% targeting efficiency?)

Answer: Thanks for your suggestion. Fig 2D has been deleted, and a trend line has been incorporated into Figure 2C.

-Throughout the paper the Myh9 introns and exons are capitalized and written both with and without a space before the number (e.g. Exon2, Exon 2, Intron2, Intron2, etc). Intent is obvious, but it should be at least consistent within the manuscript. Decision to capitalize intron and exon is questionable, but not a scientific issue.

Answer: Thanks for your correction. We have used exon2 or intron2 instead of other ones in the whole manuscript to make sure the consistency.

Reviewer #3: Tan et al. have removed the comparison of the Myh9 locus from multiple species, and according to reviewers’ suggestion, the authors compared the length of homologous arms and genomics elements (CG content, SSR, CpG islands, Line, SINE) of the Myh9 locus with other mouse loci (Rosa26, HPRT, and Col1a1) that have widely been used for GT to reveal characteristics potentially contributing to the high GT efficiency. They have also analyzed genomics features of the truncated homologous arms.

Answer: All the authors thank the reviewer for appreciating the improvement of the manuscript.

Comment-1 The author removed the comparison of the Myh9 locus from multiple species, however, retains the alignment of these sequences in Supplementary Figure 1, and it may more preferably to removed Supplementary Figure 1 together.

Answer: Thanks a lot for your suggestion, Supplementary Figure 1 has been removed.

Comment-2 Without GT efficiency data from other mouse loci with different ratio of homologous arms, it is inappropriate to infer 2:1 ratio for the length of the 5’ and 3’ homologous arms facilitate GT efficiency from the result of comparison the Myh9 locus with other loci.

Answer: Thanks a lot for your consideration, we have amended this speculation for other loci.

Comment-3 As other reviewer’ suggestion, Figure 3C is basically Figure 3D, the authors should only present Figure 3D and removed Figure 3C. The concern remaining in the Figure 3D, as targeting efficiency will not be lower than 0, it is more appropriate to set the minimum value of the vertical axis to 0.

Answer: Thanks a lot for your suggestion. Fig 2C and Fig2D has been incorporated into one, e.g Fig2C in the revised version, your concern has also been solved.

Comment-4 In Figure 2A, No.2 total length is 4.6 kb, and the right homologous arm of No.3 should be left aligned.

Answer: Thanks a lot for your correction, this error for construct No2 has been amended, while the information for construct No3 was right, because we deleted the exon2 and beyond (an about 0.5kb) to obtain this left truncated form of 3’arm. A note for this has been added in the legend for Figure2A.

Submitted filename: Response to reviewers.doc

Click here for additional data file.

24 Feb 2020

Investigation of the molecular biology underlying the pronounced high gene targeting frequency at the Myh9 gene locus in mouse embryonic stem cells

PONE-D-19-15918R3

Dear Dr. Wang,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Wenhui Hu, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

11 Mar 2020

PONE-D-19-15918R3

Investigation of the molecular biology underlying the pronounced high gene targeting frequency at the Myh9 gene locus in mouse embryonic stem cells

Dear Dr. Wang:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Wenhui Hu

Academic Editor

PLOS ONE