CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants.

Genomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish, Amphiprion ocellaris. Microinjection of zygotes was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (RH2B) involved in vision, and Tyrosinase-producing gene (tyr) involved in the production of melanin. Analysis of the sequenced target gene regions in A. ocellaris embryos showed that uptake was as high as 73.3% of injected embryos. Further analysis of the subcloned mutant gene sequences combined with amplicon shotgun sequencing revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in F0 A. ocellaris embryos. Moreover, we clearly show a loss-of-function in tyr mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful starting point to further explore the potential application of CRISPR/Cas9 in A. ocellaris, as a platform for studying gene function in anemonefishes and other reef fishes.

Introduction

Targeted genome modification (i.e., reverse genetics) is an elegant approach for directly attributing genotype with phenotype but has been limited in non-model organisms owing to a lack of high-quality assembled genomes, affordable technologies, and species-specific protocols. Modern gene-editing tools such as the clustered-regularly-interspaced-short-palindromic-repeat (CRISPR/Cas9) system enables precise targeted gene-editing, where a synthetic guide RNA (sgRNA) directs the cutting activity of Cas9 protein to produce a double strand break at a genetic location of interest. Subsequent error prone DNA repair by non-homologous end joining (NHEJ) often leaves insertions and/or deletions (indels), which may induce a frameshift and potential loss of gene function [1]. The injection of sgRNA fused with Cas9 protein has proven to be an effective tool for precise genome editing at target gene sequences in the cell lines of numerous species including many teleost fishes such as zebrafish (Danio rerio) [2], Nile tilapia (Oreochromis niloticus) [3,4], medaka (Oryzias latipes) [5], Atlantic salmon (Salmo salar) [6], killifish (spp.) [7,8], pufferfish (Takifugu rubribes) [9,10], and red sea bream (Pagrus major) [11]. However, the CRISPR/Cas9 system has yet to be applied to coral reef fishes, a highly diverse assemblage of species with a unique life history and biological adaptations suited for survival in their reef environment (e.g., a pelagic larval stage, demersal spawning, and parental behaviour) [12-14] but make them incompatible with standard CRISPR protocols used on most teleosts. Thus, requiring the development of a new approach.

One such group of reef fishes are anemonefishes (subfamily, Amphiprioninae), an iconic group that shelter in certain species of sea anemones [15], and are sequential hermaphrodites [16,17] that live in strict social hierarchies determined by body size [18]. The fascinating aspects of anemonefish biology has led to their use in multiple areas of research including for studying the physiological responses of reef fishes to the effects of climate change [19-21], the hormonal pathways that regulate sex change [22,23] and parental behaviour [24-26], and the physiological adaptations for group-living [18,27]. Moreover, anemonefishes are being used to understand the visual capabilities of fish [28,29] and evolution of skin colour diversity [30-32] in reef fishes. However, despite the wide-reaching applications of anemonefish research, the genetic basis for many of their traits remain to be empirically investigated. Consequently, anemonefish studies have been limited to correlative findings from comparative transcriptomics [30-32] and/or indirect comparisons by using reverse genetics/testing genetic elements of interest in pre- established models such as zebrafish [32]. Recently, the release of assembled genomes for multiple anemonefish species [33-35] has made it feasible to apply the CRISPR/Cas9 system to conduct genome modification in anemonefishes.

Producing biallelic knockout animals within the first generation (F0) is often desirable in species with long generation times where the establishment of stable transgenic lines might take several years. This is true for anemonefishes which have a relatively long development time till reproduction (~12–18 months), and therefore, are poorly suited for studies that rely on multigenerational breeding schemes to generate results. Thus, a well-designed protocol for the efficient delivery of sgRNA/Cas9 to completely knockout gene function is needed. To achieve this, careful species-specific considerations must be made for sgRNA design, dose toxicity, construct delivery parameters (i.e., air pressure, needle dimensions), and egg/embryo- care both during microinjection and incubation [10]. Inherent challenges specific to gene-editing anemonefishes and many other demersal spawning reef fishes include the injection and/or care of their substrate-attaching eggs [36] and pelagic larval stage [37]. Therefore, a protocol for performing CRISPR/Cas9-mediated genome editing in anemonefishes would be highly beneficial for diverse areas of research to directly test candidate genes facilitating e.g., sex change [23], colour vision [29] and skin pattern development [32].

In this study, we describe a protocol for performing CRISPR-Cas9 in the false clown anemonefish, Amphiprion ocellaris, an ideal species for gene-editing due to the public availability of its long-read assembled genome [33], its relative ease of being cultured in captivity [38] and being the most widely studied anemonefish species [39]. This has led the community to work on adapting a CRISPR/Cas9 approach simultaneously [40]. To demonstrate our protocol, we report on its efficacy in producing biallelic knockouts in F0 A. ocellaris embryos. Newly fertilised embryos were injected with a construct of synthetic guide RNA and recombinant Cas9 protein that separately targeted two genes: The rhodopsin-like 2B opsin gene (RH2B) encoding a mid-wavelength-sensitive visual pigment [41], and the Tyrosinase encoding gene (tyr) involved in the initial step of melanin production [42]. Moreover, genomic sequencing and skin (melanism) phenotypes revealed in many individuals a complete loss of gene function. We hope this protocol provides a useful resource for future gene-editing experiments involving anemonefishes and other demersal spawning reef fishes.

Materials and methods

Care and culturing of A. ocellaris

Captive-bred pairs of A. ocellaris purchased from a local commercial breeder (Clownfish Haven, Brisbane Australia) were housed in recirculating aquaria at The Institute for Molecular Bioscience at The University of Queensland, Australia. Experiments were conducted in accordance with Animal Ethics Committee guidelines and governmental regulations (AEC approval no. QBI/304/16; Australian Government Department of Agriculture permit no. 2019/066; UQ Institutional Biosafety approval no. IBC/1085/QBI/2017). Individual aquaria (95 L) contained a single terracotta pot (27 cm diameter) that provided a shelter and egg-laying structure for brood-stock fish. Spawning usually occurred during the late-afternoon to evening (15:00–18:00), which was preceded by a fully protruded ovipositor and behaviours that included surface cleaning and ventral rubbing on pot surfaces. Eggs laid by the female were adhered to the pot and subsequently fertilised by the male (Fig 1). Because injected eggs are rejected by parents after being returned, we incubated the eggs in an isolated tank (36 L) which contained heated (26°C) marine water (1.025 sg) dosed with methylene blue (0.7 mL, Aquasonic), and kept aerated using a wooden air diffuser (Red Sea). Dead eggs/embryos were removed daily to minimise the risk of fungal or other disease outbreaks.

Fig 1

Egg microinjection site and embryo appearance.

(A) Illustration depicting an Amphiprion ocellaris egg (< 1-hour post-fertilisation) with demarcated injection site at the animal pole. (B) Brightfield micrograph of a live A. ocellaris egg injected by a microneedle with released fluid marked by red-fluorescent dye. (C) Wild-type A. ocellaris embryo at 64–88 hours post-fertilisation with formed eyes and pigmentation.

Six to seven days post-fertilisation, the eyes of embryos visibly silvered, and they were ready to hatch. Because larvae in our system (both mutant and wildtype) often struggled to hatch properly despite being provided optimal external conditions (e.g., no-light, warmth, water motion), we resorted to using a non-standard approach, where larvae were manually hatched. Eggs which contained larvae were viewed under a microscope while immersed, and a small pair of dissection scissors were used to make an incision near the base of the egg on its substrate-attaching side, and then a pair of fine-tipped forceps were used to gently pry the chorion apart to produce a large enough hole for the larva to emerge. Free-swimming larvae were then immediately transferred to a grow-out tank (35x20x35 cm) and raised following a standard anemonefish rearing approach [for more details and alternative protocols see 43]. Larvae were kept in an aquarium with sides wrapped in black plastic that eliminated all horizontal light to prevent bodily damage from repeated swimming into the tank walls. Tank water was kept circulated using a low flowrate air pump with stone. Live rotifers (Brachionus spp.) were introduced at a high dosage (~10 rotifers/mL) as a food source, along with microalgae (Nannochloropsis spp.) which tinted the water green. 24-hrs post-hatch (i.e., 1 dph), a very dim overhead (single blue LED strip) light on a 12:12 hour timer was introduced to encourage feeding while not stressing the larvae. Rotifer density was maintained till 6 to 7 dph, after which freshly (24-hr old) hatched nauplii of Artemia spp. were introduced. By 10 dph, the diet of anemonefish larvae was fully transitioned to exclusively Artemia (~3 nauplii/mL). An air-sponge filter was installed 10 dph to control ammonia levels, and overhead lighting was changed to a slightly brighter white light. An artificial diet of pellets (75–250 μm) was introduced 14 dph, which coincided with the completion of metamorphosis. Juveniles (~30 dph) were transitioned to larger pellets (500–800 μm, Ocean Nutrition), by which point fish were approximately 3.0 cm in standard length.

Design and in-vitro testing of sgRNAs

To trial the application of the CRISPR-Cas9 system in anemonefishes, we designed four and two sgRNAs that targeted A. ocellaris RH2B and tyr genes, respectively (Fig 2A and 2B). The gene sequence for A. ocellaris RH2B was obtained from a previous study [29], and the same approach described by Mitchell et al. 2021 was used to identify the tyr gene sequence in the A. ocellaris genome [33]. All gene sequences were viewed in Geneious Prime (v.2019.2.3, https://www.geneious.com/), and the “Find CRISPR Sites…” function was used to screen suitable sgRNA sequences with search parameters that included a target sequence length of 19-bp or 20-bp, an ‘NGG’ protospacer-adjacent-motif (PAM) site located on the 3’ end of the target sequence (see Supporting Information S1 File for a list of sgRNA sequences). All selected target sequences were scored for their off-target activity compared against the A. ocellaris genome using an inbuilt scoring algorithm implemented in Geneious and originally designed by Hsu et al. (2013) [44]. Each off-target site is given a score based on how similar it is to the original CRISPR site and where any mismatches occur (i.e., mismatches near the PAM site will affect binding more than those further away from the PAM site). A higher score for an off-target site indicates a higher similarity to the original CRISPR site, and a higher likelihood of the sgRNA/Cas9 binding to the off target. The overall specificity score for a CRISPR site is calculated as 100% minus a weighted sum of off-target scores in the target genome. Thus, a high specificity score indicates a more ideal CRISPR site with few or weak potential off targets. We screened and selected sgRNAs with no major off-target sites (overall specificity score ≥90%). Both the sgRNAs and purified Cas9 protein fused with nuclear-localisation-signal (NLS) used in this study were purchased from Invitrogen (catalogue no. A35534, A36498; https://www.thermofisher.com/). One forward-directed cutting sgRNA on the RH2B gene targeted a sequence on Exon 4 immediately upstream (18-bp) of the conserved chromophore binding site Lys296 [45], where a frameshift would prevent the formation of a functional visual pigment. To assess cutting activity at other RH2B sites, we selected three additional target sequences, including one on Exon 1, and two on Exon 5 (i.e., downstream of Lys296). Two sgRNAs targeted sites on Exon 2 of the tyr gene, a location adequately upstream where reading frame shifts produced by indel mutations would more likely knockout gene function, while being far enough downstream to reduce the likelihood of alternative transcription start sites being utilised. The cutting activity of our sgRNAs with Cas9 were initially assessed in-vitro by incubating PCR amplicons of targeted gene regions with or without sgRNA and/or Cas9 and comparing fragment length via gel electrophoresis (see Supporting Information S2 File for full details on PCR routine, and in-vitro assay reagent quantities and incubation parameters) (Fig 2C and 2D).

Fig 2

Targeted gene regions and in-vitro cutting assay.

Sites and sequences targeted by sgRNA designed for the (A) RH2B and (B) tyr genes in Amphiprion ocellaris. Expanded regions show the target sequence (underlined in green) and ‘NGG’ PAM (underlined in black) for each sgRNA. For Exon 4 of RH2B, the Lys296 chromophore binding site (coloured blue) is also depicted down-stream of target sequence 1. Gel images to the right of each gene illustration depict DNA fragments size when amplicons that contained target (C) RH2B and (D) tyr gene regions were incubated (in-vitro) with (+) or without (-) Cas9 protein and sgRNA. Dotted boxes highlight cut DNA fragments.

Microinjection delivery of CRISPR-constructs

The clutches were collected 10–15 minutes post-fertilisation for CRISPR-construct delivery to ensure adequate fertilisation of eggs but before the first cell division had occurred 60–90 min post-fertilisation [46]. Both before and during injecting, pots containing egg clutches were broken apart into multiple shards (~2.0x4.0 cm) using a hammer and chisel. The shards were then placed in a petri dish and partially submerged in Yamamoto’s ringer’s solution [47] (see Supporting Information S3 File) to alleviate osmotic stress associated with injection [10]. Eggs were viewed under a dissection microscope (3.5x magnification) and microinjected directly into the animal pole (Fig 1A and 1B) at a 45° angle with a pulled borosilicate glass pipette (Harvard Apparatus: 1.0x0.58x100 mm) fitted on a pneumatic injector unit (Narishige IM- 400) and micromanipulator (Marzhauser MM3301R). Injector pressure settings were configured to deliver a 1 nL dose of a mixture per egg. Our initial mixture contained sgRNA (200 ng/μL, 13.8 μM), Cas9 protein (500 ng/μL, 12.3 μM) and KCl (300 μM), that was suspended by slowly pipetting up-and-down in a 10 μL stock-solution containing 5.5 μL RNAse free H2O and incubated at 37°C for 10 minutes to form a sgRNA/Cas9 construct then stored on ice, 20–30 min before injections started. Both the inclusion of KCl solution to aid in sgRNA/Cas9 mix solubility, and incubation step were adapted from Burger et al. (2016) [48]. 2 μL of the solution was then backloaded into a microneedle immediately before injection (see Supporting Information S4 File for details on microneedle dimensions and injector pressure settings). Injecting ceased when the chorion had become too thick to penetrate (~40–50 minutes post-fertilisation). To assess the mortality attributed to toxicity of the injection dosage and damage induced loss, the survival rate of CRISPR-Cas9 injected eggs were compared to controls, including: 1) eggs injected with a mixture containing no Cas9 (replaced with water), 2) non-injected eggs, and 3) a mixture containing diluted sgRNA (5 μM) and Cas9 protein (5 μM).

To control for differences in individual user, we had multiple personnel perform injections across clutches. Survival rates were calculated as the proportion of live embryos (Fig 1C) at collection relative to the number of embryos per treatment at <1-hour post-fertilisation (hpf).

Genotype and phenotype analysis of mutants

Treatment and control embryos were collected 64–88 hours post-fertilisation when eyes were clearly visible (Fig 1C). Tissue samples were taken as fin-clips from juveniles at about three months post-hatch. DNA was extracted from embryos and fin-clips using a DNeasy Blood & Tissue kit (Qiagen catalogue no. 69504), as per the manufacturer’s protocol. The concentration and purity of the extracted DNA was first tested via Nanodrop (IMPLEN N60) and then PCR-amplified using primers flanking the targeted gene location (see Supporting Information S2 File for primer sequences). Sanger sequencing of PCR amplicons was outsourced to AGRF (https://www.agrf.org.au/) and positive mutants were detected by mapping their sequences against the respective gene in Geneious. Because all positive mutants had a degree of mosaicism, we identified the full range of mutations by subcloning the PCR products of four RH2B (clutch no. 3) and four tyr (clutch no. 12) mutant embryos from clutches with high somatic activity using the Invitrogen TOPO TA kit according to the manufactures protocol (Invitrogen catalogue no. K4575J10), and Sanger sequenced the extracted plasmids from 6–10 colonies per sample. This process was also performed using fin-clips taken from three-month-old RH2B mutant juveniles (n = 3) from clutch no. 16.

To further analyse the mosaicism of our mutagenesis approach, we submitted two samples per target gene for next generation shotgun amplicon sequencing (NGS) to Novogene (https://en.novogene.com/) using Illumina NovaSeq paired-end sequencing with insert sizes of 150bp for RH2B (1 Gbp) and 250bp for tyr (1 Mbp). Raw reads were processed in Geneious by first trimming adapters and low-quality bases (phred scores <20) from the end of reads using the ‘BBDuk’ plugin (v.38.84; https://www.geneious.com/plugins/bbduk/), and then merged paired reads using the tool ‘BBMerge’. Next, we used the ‘Analyse CRISPR Editing Results’ tool in Geneious which mapped merged reads to an unedited reference sequence (50bp sequence forward and reverse of the CRISPR site), then collapsed identical variants (≥0.04% minimum variant frequency) and returned a number of reads as a percentage of the total mapped reads.

Brightfield micrographs were taken (Nikon SMZ800N) of individual tyr mutant embryos and a wildtype embryo for comparison.

Results and discussion

sgRNA in-vitro assay

An in-vitro assessment of sgRNA cutting activity was conducted to verify the integrity and viability of our sgRNA designs with target sites located on either A. ocellaris RH2B opsin gene (Fig 2A) or tyr gene (Fig 2B). All five selected sgRNAs exhibited positive cutting activity after incubation with amplicons that encompassed the target regions (Fig 2C and 2D). Cutting activity indicated the sgRNA designs were suitable for in-vivo trials. No cutting activity was observed when amplicons were incubated without sgRNA (for tyr) or Cas9 (for RH2B).

Survival and mutation rate

Overall, negative control or non-injected clutch survival ranged between 25.7% to 93.6% (mean ± sd = 62.9 ± 19.0%) and was consistently higher than survival of sgRNA/Cas9 injected embryos, which ranged from 12.5% to 48.3% in RH2B targeted embryos (29.2 ± 9.0%), and 16.3% to 27.7% in tyr targeted embryos (22.2 ± 4.8%) (Tables 1 and 2). However, inter-clutch survivability was overall highly variable, a possible consequence of variable broodstock quality and/or experience levels in spawning. Survival of positive control (sgRNA-only injected) embryos ranged between 26.1% to 73.7% (45.4 ± 17.4%) (Tables 1 and 2), and no clear improvement was detected when eggs were injected with a >50% reduced concentration of sgRNA/Cas9 (33.0 ± 10.8%) (in clutches 8–11; Table 1). These observed differences in survival between the injected treatments and (non-injected) control embryos, indicated that physical trauma from the injection process was most likely the main contributor to mortality observed in injected embryos. A reduction in needle tip-size (<15 μm) may help lower mortality; however, in our experience thinner needles exhibited excessive bending when attempting to penetrate the thick chorion of anemonefish eggs. Only needles with a relatively short-taper and broad tip (i.e., stubby profile) were usable for injections. Natural thickening of the chorion peaked at 30–40 minutes post-fertilisation (about 50–60 minutes preceding the first cell division) and prohibited further injecting regardless of needle size.

Table 1

Clutch survival and mutation rates for RH2B targeted injection rounds.

Survival rates of embryos injected with sgRNA/Cas9 (treatment) or sgRNA-only (positive control) targeting RH2B, and non-injected (negative control) embryos at time of collection (64–88 hpf), number of genotyped embryos, and mutation rate per clutch and target sequence. Note: Clutches 8–11 (sgRNA 4) were injected with a lower concentration of sgRNA/Cas9.

Clutch no.	1	2	3	4	5	6	7	8	9	10	11
RH2B sgRNA	1	1	1	2	2	3	3	4	4	4	4
Injected survival	11/22	14/48	13/48	22/75	7/56	17/50	16/45	75/147	64/277	43/141	102/340
64–88 hpf	22.0%	29.2%	27.1%	29.3%	12.5%	34.0%	35.6%	48.3%	23.1%	30.5%	30.0%
No. of genotyped (out of injected)	11/11	13/14	13/13	22/22	6/7	17/17	9/16	15/75	-	-	7/19*
Positive mutants	1/11 9.1%	3/13 23.1%	4/13 30.8%	0/22 0%	1/6 16.7%	8/17 47.1%	3/9 33.3%	11/15 73.3%	-	-	4/7*
Non-injected survival rate 64–88 hpf	25/59 42.4%	40/50 80.0%	25/42 59.5%	14/26 53.8%	16/52 30.8%	44/47 93.6%	51/61 83.6%	47/80 58.8%	71/92 77.2%	-	-
Injected (sgRNA-only) survival 64–88 hpf	-	-	-	-	4/10 40.0%	5/12 41.7%	-	-	-	-	-

hpf, hours post-fertilisation.

*proportion of positive mutants identified by fin-clip extractions taken from juveniles.

Table 2

Clutch survival and mutation rates for tyr targeted injection rounds.

Survival rates of embryos injected with sgRNA/Cas9 or sgRNA-only targeting tyr, and non-injected embryos at time of collection (64–88 hpf), number of genotyped embryos, and mutation rate per clutch and target sequence.

Clutch no.	12	13	14	15	16
tyr sgRNA	1	1	2	2	2
Injected survival	14/86	74/267	9/47	39/148	27/126
64–88 hpf	16.3%	27.7%	19.1%	26.4%	21.4%
No. of genotyped (out of injected)	13/14	74/74	8/9	39/39	27/27
Positive mutants	7/13 53.8%	9/74* 12.2%*	2/8 25.0%	7/39* 17.9%*	12/27* 44.4%
Non-injected survival rate 64–88 hpf	37/45 82.2%	19/74 25.7%	3/10 30.0%	52/67 77.6%	57/67 85.1%
Injected (sgRNA-only) survival 64–88 hpf	-	-	-	12/46 26.1%	14/19 73.7%

hpf, hours post-fertilisation

*proportion of positive mutants identified solely by hypomelanistic phenotype out of all surviving embryos.

Examination of the target gene sequences of injected embryos showed highly variable mutation rates that ranged from 0% to 73.3% for RH2B (n = 10 clutches; Table 1), and 12.2% to 53.8% for tyr (n = 5 clutches; Table 2). In RH2B targeted fishes raised till the juvenile-stage (clutch 11; Table 1), we found a relatively high mutation rate of 57.1%. We also found that lowering the injected sgRNA/Cas9 concentration (<50%) had no apparent impact on mutation rate (clutch 8 = 33.3%, clutch 11 = 57.1%; Table 1). To achieve higher mutation rates, we suggest a couple alternative options such as by improving the accuracy of injecting the animal pole by delaying injection until the formation and visible swelling of the blastodisc (~40–50 minutes post-fertilisation) that precedes the first cell division; however, this severely limits the number of injectable eggs due to thickening of the chorion. Alternatively, the substitution of Cas9 protein with Cas9 mRNA may circumvent the need for direct delivery into the nucleus and permit injection elsewhere (e.g., in the yolk). Although Cas9 protein has been associated with a higher efficiency of mutagenesis than Cas9 mRNA [49], the relatively long-lived (~90 minutes) single cell stage of the A. ocellaris zygote [46] would likely permit adequate time for migration into the nucleus and translation processes. The incorporation of NLS-fused Cas9 mRNA could also help compensate for differences in uptake efficiency [50].

Genotype analysis of mutants

Analysis of the subcloned sequences of RH2B (clutch 3, RH2B 1; Fig 3A) and tyr (clutch 12, tyr 1; Fig 3B) mutant A. ocellaris embryos, revealed that our approach was successful in producing biallelic mutations in seven out of the eight embryos; only one tyr mutant retained a wildtype allele. This high (75% to 100%) efficiency in inducing biallelic mutations in F0 A. ocellaris proves promising for the use of reverse genetics in animals with long generation times (12–18 months in the case of anemonefishes) [51], allowing experiments to start while waiting for stable homozygous-lines to be established. Although verifying germline transmission in F0 brood-stock will be required for long-term, inter-generational studies.

Fig 3

Genotype analysis of RH2B mutant Amphiprion ocellaris embryos.

Subcloned sequences and next generation shotgun amplicon sequences (NGS) belonging to A. ocellaris embryos (clutch 3, sgRNA RH2B 1; clutch 12, sgRNA tyr 1) with mutations at targeted sequences (underlined) located on (A) Exon 4 of the RH2B opsin gene, and (B) Exon 2 of the tyr gene. Wildtype (WT) sequences are included for reference. Mutations included deletions (dashes), substitutions (green), and insertions (blue). Sequence labels on the left-side indicate mutant embryo and allele no., while numbers on the right-side indicate the base pair change (Δbp), proportion of each allele out of the total number of cloned sequences for each embryo, or the percentage (%) of reads out of total reads for NGS. (C) Number of frameshift and in-frame mutations per RH2B mutant embryo. (D) Micrographs of tyr mutant A. ocellaris embryos exhibiting full knockout (tyr-M1 and -M2) and partial knockout (tyr-M3 and -M4) phenotypes, and a wildtype embryo for comparison. (E) Number of frameshift and in-frame mutations per tyr mutant embryo.

A total of 24 and 11 distinct mutations were found in RH2B mutants (Figs 3A and 4A) and in tyr mutants (Fig 3B), respectively. Although most mutations were detected by both the sequencing of subcloned colonies and NGS (see Supporting Information S5 File for full details on all variants detected by NGS), the greater sampling depth of the latter (total no. of reads: RH2B-M1 = 3649143, RH2B-M4 = 3518312, tyr-M2 = 83196, tyr-M4 = 664560) revealed additional mutations in RH2B-M1 (n = 4), RH2B-M4 (n = 2), tyr-M2 (n = 1), and tyr-M4 (n = 4). Most mutations were in the form of deletions that ranged in length between 1 – 43bp, while fewer insertions ranged from 1–10 bp. An extremely large deletion of 449bp was detected in RH2B-M5 and RH2B-M6 (Fig 4A). Mutations were situated (4 – 14bp) upstream (‘5) of their respective PAM sequence, a proximity and location typically reported for Cas9 cutting activity [52] (Fig 3A and 3B). Exceptions included deletions starting at the PAM in tyr-M2 and tyr-M3 (-7bp), and that spanned regions both up- and down-stream of the PAM in RH2B-M4 (-43bp), RH2B-M5 and RH2B-M6 (-449bp). The most frequent mutations found in multiple RH2B mutants included a 5bp deletion (10bp upstream of PAM) and a 2bp deletion (14bp upstream of PAM) (Fig 3A), while the most common mutations across tyr mutants were a 1bp deletion (4bp upstream of PAM) and a 7bp deletion (starting at PAM) (Fig 3B). Both RH2B (Figs 3A and 4A) and tyr (Fig 3B) mutant embryos had between two to seven distinct mutations. This high number of mutations per embryo suggests Cas9 cutting activity persisted beyond initial cell division, an indication of a high dosage of sgRNA and Cas9, that could potentially be reduced further if desired.

Fig 4

Genotype analysis of four-month-old RH2B mutant Amphiprion ocellaris.

(A) Subcloned sequences belonging to A. ocellaris juveniles (clutch 11, sgRNA RH2B 4) with mutations at targeted sequences (underlined) located on Exon 1 of the RH2B opsin gene. Wildtype (WT) sequence is included for reference. Mutations included deletions (dashes), substitutions (green), and insertions (blue). Sequence labels on the left-side indicate mutant fish and allele no., while numbers on the right-side indicate the base pair change (Δbp) and the proportion of each allele out of the total number of cloned sequences for each fish. (B) Images of the RH2B mutant A. ocellaris juveniles. (C) Number of frameshift and in-frame mutations per RH2B mutant fish.

Analysis of the subcloned sequences of RH2B mutant juveniles (from clutch 11, RH2B 4; Fig 4A), showed biallelic mutations in two out of the three fish examined (Fig 4B). Only one juvenile (M7) was found to possess a WT allele, along with two in-frame mutations (Fig 4A and 4C). Both juveniles M5 and M6, were found to possess only frameshifted sequences (Fig 4C), which also likely have impaired RH2B gene function. This further demonstrated the long-term viability of mutants produced using our CRISPR/Cas9 approach.

Interestingly, analysis of NGS data revealed an identical 38bp deletion in both examined tyr mutants (tyr-M2.5 and tyr-M4.6; Fig 3B), which spanned the entirety of the CRISPR site and PAM. This mutation was found by the majority of mapped reads in mutants tyr-M2 (59.6%; n reads = 49608) and tyr-M4 (99.1%; n reads = 658075), that is highly unusual when considering that it went completely undetected by the subcloned sequencing analysis of both embryos. Moreover, the location of this mutation is atypical of double-stranded breaks induced by CRISPR/Cas9 [52]. A second NGS run returned similar results, and therefore, it did not appear to be a sequencing or library preparation error. We suggest this deletion was possibly an artefact from PCR during sample preparation, rather than a genuine mutation.

Because there were no easily discernible phenotype(s) in RH2B mutant embryos, we speculate on the loss of gene function based on the frameshift or in-frame nature of mutations (Figs 3C and 4C). Four of the seven subcloned RH2B mutants (RH2B-M1, -M4, -M5, -M6) possessed a full complement of mutant alleles that exhibited frameshifts (Figs 3 and 4). Examination of the translated (frameshifted) sequences (Supporting Information S6 File for an alignment of translated sequences) confirmed the presence of missense mutations that disrupted the chromophore binding site (Lys296), and downstream premature stop codons that may preclude visual pigment formation. Thus, it is likely these four embryos and fish had/have either a complete knockout or at least impaired RH2B gene function. Future attempts to remove the entire chromophore binding site could involve co-injecting upstream and downstream positioned sgRNA.

Phenotype analysis of mutants

CRISPR/Cas9 knockout of A. ocellaris tyr produced embryos which exhibited varying degrees of hypomelanism (Fig 3D), a phenotype attributed to the disruption of the enzymatic conversion of tyrosine into melanin and is similarly observed in tyr knockout zebrafish embryos and larvae [53,54]. In comparison, wildtype A. ocellaris embryos consistently had heavily pigmented skin and eyes. A complete lack of melanin was observed in two (tyr-M1 and tyr-M2) out of the 14 injected embryos from clutch 12 (Fig 3D). Analysis of their subcloned sequences and NGS data revealed both had biallelic mutations, all of which are likely to induce frameshifts that render TYR non-functional (Fig 3E). Whereas partial depigmentation or a mosaic appearance was found in five out of the 14 embryos (e.g., tyr-M3 and tyr-M4; Fig 3D), most likely as a result of an incomplete knockout of TYR activity caused by in-frame mutations (tyr-M3.1, 3.2, 3.5, and tyr-M4.7; Fig 3B and 3D) and/or wild type alleles (tyr-M4.3; Fig 3B). The nature of this skin pigmentation phenotype has been shown in zebrafish to be sgRNA/Cas9 dose- dependent [54]; however, in our case the nature of the mutation (i.e., in-frame or out-of-frame) was also a major determinant of phenotype. Notably, no WT allele was detected by NGS in tyr-M4 despite being found as a subcloned sequence (tyr-M4.3; Fig 3B), it is unclear what may have caused this discrepancy.

Behavioural experiments will be necessary to demonstrate a functional loss of visual opsin in RH2B mutant anemonefish, as has been demonstrated in opsin knockout strains of medaka that exhibit impaired spectral sensitivity in optomotor tests [55] and/or altered social behaviour [56,57]. Applying this same approach to other visual opsin genes could also help attribute the input of different visual pigments to vision (e.g., in colour and/or brightness perception). Similarly, the loss of TYR could also be assessed for its impact on colour sensitivity, as has been reported in zebrafish [58].

Conclusions and further directions

Here we present the first use of the CRISPR/Cas9 system in a reef fish. Targeting the coding regions of the RH2B opsin and tyr genes successfully induced indel mutations in up to 73.3% of A. ocellaris embryos. Moreover, the analysis of subcloned sequences showed our gene-editing approach was able to produce biallelic mutations with an extremely high efficiency of ~90%, causing loss-of-function mutations in a substantial proportion of F0 tyr mutants. Our proven application of this technology greatly facilitates the use of CRISPR/Cas9 for a variety of other genetic applications including making precise (knock-in) gene insertions in anemonefish; however, this would require significant modification of the sgRNA to utilise homologous recombination or alternative strategies [59]. The precision of both gene knock-in and knockouts using CRISPR/Cas9 in anemonefishes could possibly benefit from applying microhomology-mediated end-joining (MMEJ) to exploit short microhomologies flanking a target site to more precisely direct cutting activity [40,60]. Combining our protocol with the latest advancements in anemonefish egg-care and larval rearing techniques [40,43], will be key in improving survival to study genome-editing in adult anemonefish. Regardless, this raises an exciting future prospect of conducting genome-editing in A. ocellaris to study the genetic basis of various unique traits in a reef fish.

sgRNA sequences.

List of injected sgRNA sequences.

(DOCX)

Click here for additional data file.

PCR details and sgRNA in-vitro assay.

Primer sequences and PCR routine, and in-vitro cutting assay reagents and incubation steps.

(DOCX)

Click here for additional data file.

Yamamoto’s ringer’s solution.

List of reagents and quantities used to make a salt-balanced solution for eggs.

(DOCX)

Click here for additional data file.

Injection parameters and configuration.

Microcapillary settings and pneumatic microinjector settings.

(DOCX)

Click here for additional data file.

Full summary of NGS data.

(DOCX)

Click here for additional data file.

Translated alignment of RH2B sequences.

Translated sequence alignment of frameshifted alleles found in RH2B-M1 and -M4, and wildtype (WT) RH2B for reference. Sequences were aligned against bovine rhodopsin (RH1) (NCBI accession no. NP_001014890.1), as an opsin template. The chromophore binding site (bovine RH1 AA no., Lys296) is boxed in blue. Amino acid (AA) numbering schemes were according to WT RH2B (upper) and bovine RH1 (lower). Translated sequences were aligned using MAFFT Alignment (v7.450) in Geneious. MAFFT reference: Katoh, K., & Standley, D. M. (2013). MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution, 30(4), 772–780. https://doi.org/10.1093/molbev/mst010.

(DOCX)

Click here for additional data file.

(PDF)

Click here for additional data file.

13 May 2021

PONE-D-21-10870

CRISPR/Cas9-mediated generation of biallelic G0 anemonefish (Amphiprion ocellaris) mutant embryos

PLOS ONE

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The study is well planned. Authors have to provide more detailed methodology and also validation of the successful knockouts, which are very much critical for this kind of manuscripts.

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Additional Editor Comments:

Authors have to provide more detailed methodology and also validation of the successful knockouts.

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Reviewer #3: Yes

**********

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Reviewer #3: Yes

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Reviewer #1: The manuscript by Mitchell et al. explore the potential application of CRISPR/Cas9 targeting in Amphiprion occellaris (anemonefish). The authors demonstrate the successful targeting of rhodopsin-like 2B opsin encoding gene (RH2B), involved in vison and Tyrosinase encoding gene (tyr), involved in the production of melanin. They show clear loss of function in tyr mutant embryos and associated phenotype. Based on this the authors suggest that CRISPR/Cas9 tool can be used for gene-editing in anemonefish, thereby it would help in studying gene function. Overall its straightforward methodology study, suggesting the use of CRISPR/Cas9 application in studying gene function in anemonefish. However, the study lacks functional and behavioral studies of mutant anemonefish fish. Further, CRISPR/Cas9 system has already been used in several fish models, such as zebrafish, tilapia, and medaka (27130213, 24728957) to study gene function or to target a specific gene. As it stands—the authors should include the functional and behavioral study of mutant fish otherwise it just an adaption of the protocol in Amphiprion occellaris and offer no technical advancement over already published studies or provide new insight.

a. The authors suggest that the mortality of experimental eggs dependent on the injection process rather than sgRNA/Cas9 cytotoxicity, however, this hypothesis is not validated.

b. It’s unclear from the study if the off-target analysis is performed.

c. The authors should verify germline transmission mutant embryo as it's important to investigate long-term function study.

d. The titration of sgRNA/Cas9 doses should be performed to optimize cas9 cutting activity.

e. The authors should perform a behavioral and functional study of mutant Amphiprion occellaris to assess the long-term effect of mutation on the viability of the fish.

Reviewer #2: Reviewer Comments to Author(s):

The manuscript describes the use of CRISPR/Cas9 for genome editing in reef fish or anemonefish. While CRISPR/Cas9 mediated mutagenesis has been performed in a previous publication (1), the rational is that no protocol has detailed how to inject anemonefish to generate mosaic mutants or complete knockouts in the F0 generation.

In the manuscript (PONE-D-21-10870), the authors established a system for implementing CRISPR/Cas9 to produce genome edited reef fish. Although the authors microinjection technique affected the survivability of injected embryos, the overall success rate of CRISPR/Cas9 mediated mutagenesis was measurable. Heritability of CRISPR/Cas9 induced mutations were not detected in the injected reef fish due to the model systems long generation time constraints. However, the authors generate mosaic founder (F0) embryos that closely resemble true null mutations, at least in the case of tyr. The ability to generate F0 mutants will allow testing the functional importance of genes, although off-target effect is more common with this approach. The research presented provides the groundwork for CRISPR/Cas9 application in anemonefish.

The methods are appropriate, and the results are clear with exceptions. An overarching concern is the claim that efficient knockdown is achieved to generate a complete knockout in the F0 without clearly explaining the mutational analysis. Overall, the manuscript readability can be revised to improve the flow of the text, but there is sufficient information presented for readers to follow the rationale, procedures and glean insights into the application of the CRISPR/Cas9 system for use on reef fish and other model organisms with longer generation times.

Injected embryos are often referred to as founders or F0 and not G0.

Lines 32, 36, 103 – “Eggs” is often used to refer to one-cell stage “embryos”. In the literature, injected “eggs” and one-cell stage “eggs” are typically “embryos”.

Lines 87, 159, 161. The authors should clarify if CRISPR/Cas9 “constructs” are injected into embryos or CRISPR/Cas9 “components” such as RNA (sgRNA) and protein (Cas9).

Lines 147 – 157 Parts of text can be revised to improve the flow of the article. For example, this section contains text that can be incorporated into the results section.

Clarification regarding the generation of “knockouts” is needed. To make the claim that efficient mutagenesis is achieved to analyze the F0 for functional analysis, it must be clear how reliable the knockdown is.

In the methods section, the authors write that “all positive mutants were heterozygous” (lines 203-204). The authors go on to state that “only one tyr mutant retained a wild-type allele” (line 281) in the results section. Were positive mutants heterozygous or did the authors find that the embryos lacked wild-type sequences when assaying their sequence clones from whole embryos? What is the authors definition of “heterozygous”?

To get a real sense of how mosaic the F0s are, more than a dozen to one hundred sequences per fish should be analyzed. More clarification is needed in regard to how many clones were sequenced (See comments for lines 280 – 282 below).

Line 242 – 247, 260 – 269. Parts of text can be revised to improve the flow of the article. Some text may be moved and incorporated in the discussion section.

Line 280 – 282

The authors should include more information to justify their choice of methods and clearly demonstrate how reliable the knockdown is. I presume the authors selected the RH2B sgRNA target 1 clutch 3 embryos because they see high somatic activity (4/13, 30.8%). Is this the case?

From the 4 embryos subjected to PCR screening for the RH2B gene, those PCR amplicons were subcloned and how many clones were sequenced? Figure 3A would suggest maybe 16 clones were sequenced. Is this also the case for tyr?

According to the text, authors subcloned and sequenced tyr PCR amplicons from embryos that came from clutch 9 (line 279). However, Figure 3 described subcloned sequenced from clutch 8 (lines 316, 331).

In regard to line 308, if the data positions the authors to do so, they can write that “no wild-type sequences were detected in any of the sequenced clones (n=# of sequenced clones).” However, lines 203-204 and 281 suggest the authors may be observing compound heterozygous fish.

Line 292 – 294. A graphical presentation can be made for the indels generated. In a bar graph, a column may show how many in frame mutations and frameshift mutations were made that were deletions and another column of the insertions that were in frame or frameshift mutations.

Another type of graph to depict the distribution of CRISPR/Cas9 induced mutations is to show number of mutations on the y axis and the base pair changes (- and +) plotted on the x axis. In this graph, authors will have bars that indicate deletion range and bars that indicate insertion sizes.

Figure 2

In Figure 2C and Figure 2D, labels for which fragment is uncut and which are cleaved can be presented. The in vitro experiment is successful, but another control is to run the sgRNA alone. In Figure 2C, the large overexposed bands may be tyr1 and tyr2 sgRNAs and not cut DNA.

References:

1. Salis P, Lorin T, Lewis V, Rey C, Marcionetti A, Escande ML, Roux N, Besseau L, Salamin N, Sémon M, Parichy D, Volff JN, Laudet V. Developmental and comparative transcriptomic identification of iridophore contribution to white barring in clownfish. Pigment Cell Melanoma Res. 2019 May;32(3):391-402. doi: 10.1111/pcmr.12766. Epub 2019 Jan 29. PMID: 30633441; PMCID: PMC6483885.

Reviewer #3: The manuscript by Mitchell LJ et al. describes CRISPR-Cas 9 genome editing in anemonefish. The authors efficiently demonstrate successful editing of two separate target sites- RH2B and Tyrosinase producing gene at G0 of A.ocellaris embryos. The experiments are well designed, performed with appropriate controls and the manuscript is well written. The manuscript will be potentially important to study gene functions in other reef fishes.

Comments:

1. Can authors comment on how is the CRISPR-Cas 9 protocol described here is novel and different from previously published CRISPR-Cas 9 editing done in other fishes?

2. Can this protocol be applied to precisely delete specific nucleotides at the target site and also Knock-in specific genes?

3. The authors show loss of function phenotype in tyr KO embryos, is there a functional phenotype to demonstrate loss of R2BH in the embryos? Probably by Immunohistochemical analysis of opsin trafficking in photoreceptors?

4. Comments on any off-target/unpredicted mutations.

**********

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Reviewer #2: No

Reviewer #3: No

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13 Nov 2021

Response to the editor and reviewers

We would like to thank Dr Anbazhagan and the three anonymous reviewers for taking the time to review our manuscript and provide insightful feedback.

We have now addressed all the concerns that were raised, and a point-by-point reply can be found below. Two versions of the manuscript, one with track-changes and one without have also been uploaded.

We hope our revised manuscript meets the requirements for publication in PLOS ONE and look forward to your decision.

Yours sincerely,

Laurie Mitchell

On behalf of all authors.

Additional Editor Comments:

Authors have to provide more detailed methodology and also validation of the successful knockouts.

- These concerns have also been raised by the reviewers and we provide detailed answers about how we addressed them below.

Reviewer #1:

Reviewer #1: The manuscript by Mitchell et al. explore the potential application of CRISPR/Cas9 targeting in Amphiprion occellaris (anemonefish). The authors demonstrate the successful targeting of rhodopsin-like 2B opsin encoding gene (RH2B), involved in vison and Tyrosinase encoding gene (tyr), involved in the production of melanin. They show clear loss of function in tyr mutant embryos and associated phenotype. Based on this the authors suggest that CRISPR/Cas9 tool can be used for gene-editing in anemonefish, thereby it would help in studying gene function. Overall its straightforward methodology study, suggesting the use of CRISPR/Cas9 application in studying gene function in anemonefish. However, the study lacks functional and behavioral studies of mutant anemonefish fish. Further, CRISPR/Cas9 system has already been used in several fish models, such as zebrafish, tilapia, and medaka (27130213, 24728957) to study gene function or to target a specific gene. As it stands—the authors should include the functional and behavioral study of mutant fish otherwise it just an adaption of the protocol in Amphiprion occellaris and offer no technical advancement over already published studies or provide new insight.

- The reviewer is concerned that our manuscript provides no technical advancement over already published studies using other teleosts with the CRISPR/Cas9 platform and requested for a behaviour or functional characterisation of the targeted genes in our study.

Regarding the first concern, we understand where the reviewer is coming from in that we here present a protocol to use CRISPR/Cas9 in another fish species. However, as opposed to the classical model systems for which the technology was adapted and applied in previously (e.g., medaka, zebrafish, and tilapia), reverse genetics in coral reef fishes faces several major challenges and to the best of our knowledge, has not been attempted before. The biggest challenge is that with demersal spawning fishes such as many smaller coral reef fishes, the eggs are attached to the substrate. For that reason, we had to develop a different approach for microinjection all together i.e., injecting eggs/embryos on shards of clay pots while attached as opposed to injecting into loose eggs/embryos as done in other fish species (lines 123-125, 211-212). The chorion of anemonefish eggs also behaves different to the one found in e.g., zebrafish, and thus needle dimensions had to be adjusted. Likewise, other injection parameters such as injection pressure etc., as well as concentrations of the vector had to be adjusted. The next major challenge is to maintain the embryos, hatching and rearing of the fry. Demersal spawners such as Amphiprion ocellaris provide egg care i.e., they clean the eggs from debris, remove dead eggs, aerate them, and protect them from predation. However, injected eggs/embryos are rejected by the parents and hence, we had to develop a method to substitute parental care. This new husbandry approach is documented in detail in lines 128-140. Hence, like the CRISPR/Cas9 protocols that have been and continue to be published for other non-model organisms e.g., corals, axolotl, fathead minnow, our approach includes major advancements that warrants separate publication.

Regarding the functional characterisation of mutant fishes. We now provide details on fully developed (4-month post-hatch), healthy RH2B opsin mutant fish in our care that have been successfully reared since our initial submission (lines 374-379, Figure 4). Sanger sequencing performed on subcloned sequences of four (out of seven) mutants shows that two are full knockouts and one is a partial knockout containing the wildtype allele (Figure 4). While a full behavioural experiment (e.g., tests for colour vision deficiency) would be ideal, this is beyond the scope of the current study. The purpose of this study is to provide, for the first time, a detailed protocol for CRISPR/Cas9 manipulation in a non-model coral reef fish species. Extensive behavioural studies take considerable amount of time, and we plan to conduct such experiments in a standalone study.

We also used amplicon shot-gun sequencing on previously extracted mutant embryos (2x RH2B mutants and 2x Tyr mutants) to gain a more complete picture of the mutations present (lines 341-348; Figure 3 A, B). This shows that most mutant alleles were picked up with the cloning approach. Most importantly, full-knockout individuals as determined by cloning did not show any wild-type alleles when using this vastly increased sequencing depth. We are therefore confident that full-knockout F0 anemonefishes can be produced using our approach.

a. The authors suggest that the mortality of experimental eggs dependent on the injection process rather than sgRNA/Cas9 cytotoxicity, however, this hypothesis is not validated.

- We have since added survival rate data from further injection rounds that used a lower concentration of sgRNA and Cas9 (lines 231, 307-309, Table 1). This found no clear improvement in survival despite maintaining a high efficiency in mutation, suggesting mortality was mainly attributable to physical trauma rather than sgRNA/Cas9 cytotoxicity.

b. It’s unclear from the study if the off-target analysis is performed.

- Our methods section (lines 172-182) describes our off-target analysis when designing our sgRNA sequences, which takes advantage of the high-resolution genome assembly for this species. We have now added further detail on this off-target screening analysis. This used an inbuilt off-target screen in the ‘Find CRISPR sites’ function of the software ‘Geneious’. The application offers a number of candidate-sgRNA sequences at a predefined region of interest for the user to select. As part of the metrics given for each sgRNA there is a specificity score calculated using a renowned method developed by the Zhang Lab at MIT.

Taken from the Geneious webpage:

“Each off-target site is given a score based on how similar it is to the original CRISPR site and where any mismatches occur (mismatches near the PAM site will affect binding more than mismatches further away from the PAM site). A higher score for an off-target site indicates a higher similarity to the original CRISPR site (and thus a higher likelihood of the CRISPR/Cas complex binding to the off target). The overall specificity score for a CRISPR site is 100% minus a weighted sum of off-target scores in the target genome. Thus, a higher specificity score indicates a better CRISPR site with few or weak potential offsite targets.”

(See https://www.geneious.com/tutorials/finding-crispr-sites-r9/)

Our database scored against the assembled genome of A. ocellaris, and we only selected sgRNAs with a score of >90%, where our RH2B sgRNAs had specificity ranging from 90 – 100%, while the tyr sgRNAs both scored 100%.

Although we cannot be absolutely certain of no off-target activity, it seems extremely unlikely. Our sgRNAs certainly induced mutations at the desired locations as verified via sequencing and also phenotypic scoring for tyr mutants.

[for full details on the specificity method see Hsu et al. (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol.].

The authors should verify germline transmission mutant embryo as it's important to investigate long-term function study.

- While we recognise the importance of analysing germline transmission for long-term experiments (i.e., for establishing homozygous lines). In our case, we want to stress the importance of CRISPR/Cas9 as a means of performing rapid gene-editing experiments to ascertain gene function within the first generation of mutants and eliminating the need for multigenerational breeding schemes. Unlike model systems (e.g., zebrafish, medaka, mice), the generation time of many non-model animals prohibits long-term experiments, and as such establishing a stable germline before starting experiments is often not feasible. Anemonefish typically take ~9-18 months to reach full sexual maturity and form a stable breeding pair, whereby the males reach sexual maturity first and females usually take a minimum of 12 months to produce their first clutch. To give an idea, this would require a minimum of 36 months to achieve a single F3 screening scheme with anemonefish. One of the advantages of the CRISPR/Cas9 platform is the ability to produce knockout mutations within the first generation of individuals and does not necessitate breeding (see e.g., Fei, J.F., et la., 2018. Application and optimization of CRISPR–Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum). Nature protocols, 13(12), pp.2908-2943.). This is one of the major reasons for our development of this protocol with anemonefish, as this will enable others to perform gene-editing experiments without the need for extensive breeding schemes. To emphasise this point more strongly, we have now included a sentence explicitly stating the long generation time of anemonefish making them unsuitable for gene-editing experiments which require breeding schemes (lines 88-90). In the mid- to long-term we aim at producing stable lines for mutants of special interest to be shared with the community.

d. The titration of sgRNA/Cas9 doses should be performed to optimize cas9 cutting activity.

- Please see our answer to ‘a.’ on line 231 we describe another injection treatment using a reduced 5 µm sgRNA/Cas9 concentration, and then show that cutting efficiency was maintained (Table 1 clutches 8-11, and lines 281-283). While our current cutting efficiency is more than adequate to produce full-knockout progeny, we suggest other potential options that may improve it further (see lines 309-320).

e. The authors should perform a behavioral and functional study of mutant Amphiprion occellaris to assess the long-term effect of mutation on the viability of the fish.

- Please see our initial response. In short, while a full behavioural experiment (e.g., tests for colour vision deficiency) would be ideal, this is beyond the scope of the current study which is a protocol, as a proof-of-concept for performing CRISPR/Cas9 in a reef fish. Extensive behavioural studies take considerable amount of time, and we plan to conduct this as an entirely separate study/experiment. However, we have now included sequencing results for (three-month-old) juvenile mutants successfully raised in our hatchery system. This shows the long-term viability of our mutagenic fish.

Reviewer #2

Reviewer #2: Reviewer Comments to Author(s):

The manuscript describes the use of CRISPR/Cas9 for genome editing in reef fish or anemonefish. While CRISPR/Cas9 mediated mutagenesis has been performed in a previous publication (1), the rational is that no protocol has detailed how to inject anemonefish to generate mosaic mutants or complete knockouts in the F0 generation.

- CRISPR/Cas9 has not been attempted in anemonefishes or reef fishes before. The study the reviewer is referring to here, used zebrafish to test orthologous gene sequences for iridophore development that were originally detected in anemonefishes using a comparative transcriptomic approach.

In the manuscript (PONE-D-21-10870), the authors established a system for implementing CRISPR/Cas9 to produce genome edited reef fish. Although the authors microinjection technique affected the survivability of injected embryos, the overall success rate of CRISPR/Cas9 mediated mutagenesis was measurable. Heritability of CRISPR/Cas9 induced mutations were not detected in the injected reef fish due to the model systems long generation time constraints. However, the authors generate mosaic founder (F0) embryos that closely resemble true null mutations, at least in the case of tyr. The ability to generate F0 mutants will allow testing the functional importance of genes, although off-target effect is more common with this approach. The research presented provides the groundwork for CRISPR/Cas9 application in anemonefish.

The methods are appropriate, and the results are clear with exceptions. An overarching concern is the claim that efficient knockdown is achieved to generate a complete knockout in the F0 without clearly explaining the mutational analysis. Overall, the manuscript readability can be revised to improve the flow of the text, but there is sufficient information presented for readers to follow the rationale, procedures and glean insights into the application of the CRISPR/Cas9 system for use on reef fish and other model organisms with longer generation times.

- We recognise the need to make our mutation analysis clearer (see below for specific addressment).

Injected embryos are often referred to as founders or F0 and not G0.

- We have amended our manuscript to now refer to all injected embryos as founders or ‘F0’.

Lines 32, 36, 103 – “Eggs” is often used to refer to one-cell stage “embryos”. In the literature, injected “eggs” and one-cell stage “eggs” are typically “embryos”.

- All relevant lines have now been amended to read “embryos” for the post-fertilised, single cell stage.

Lines 87, 159, 161. The authors should clarify if CRISPR/Cas9 “constructs” are injected into embryos or CRISPR/Cas9 “components” such as RNA (sgRNA) and protein (Cas9).”

- We have now clarified in the manuscript that sgRNA/Cas9 complexes are injected. This complex is formed during the incubation period that precedes injecting (see line 222)

Lines 147 – 157 Parts of text can be revised to improve the flow of the article. For example, this section contains text that can be incorporated into the results section.

- We have moved the mention of using two guides to cut out the entire gene region to the results/discussion section (see lines 407-409). The remaining text in this section pertains to our sgRNA design and as such should remain in the methods.

Clarification regarding the generation of “knockouts” is needed. To make the claim that efficient mutagenesis is achieved to analyze the F0 for functional analysis, it must be clear how reliable the knockdown is.

In the methods section, the authors write that “all positive mutants were heterozygous” (lines 203-204). The authors go on to state that “only one tyr mutant retained a wild-type allele” (line 281) in the results section. Were positive mutants heterozygous or did the authors find that the embryos lacked wild-type sequences when assaying their sequence clones from whole embryos? What is the authors definition of “heterozygous”?

- We now recognise that our previous use of terminology was inconsistent and confusing. Now, we have amended throughout the manuscript that heterozygous mutants are those with two or more distinct alleles, as opposed to homozygous (i.e., one allele type present). In our case, all the embryos/fish are heterozygous, but only one individual contained a wild-type allele. None of our individuals showed only a single mutation. Furthermore, our NGS results for two RH2B mutants and two tyr mutants found no evidence of wildtype alleles, indicating that our knockout was reliable.

To get a real sense of how mosaic the F0s are, more than a dozen to one hundred sequences per fish should be analyzed. More clarification is needed in regard to how many clones were sequenced (See comments for lines 280 – 282 below).

We have now also provided NGS amplicon sequencing results for the two individuals (per target gene) which had the least number of subcloned sequences i.e., we sequenced hundreds of thousands to millions of sequences per individual. Variant sequences only detected via NGS are enclosed in boxes and given a percentage out of the total reads that covered the target region. While the most frequent sequences detected were all represented by the subcloning analysis, we did find a few previously undetected variants, but none were WT or represented in-frame mutations. The amplicon sequencing approach confirms that F0 full-knockout individuals can be produced using our method.

Line 242 – 247, 260 – 269. Parts of text can be revised to improve the flow of the article. Some text may be moved and incorporated in the discussion section.

- The mentioned section is a combined results/discussion. For lines 242-247, the possibility of using a smaller needle-size to possibly reduce mortality follows the results on survivorship. We believe this to be the most appropriate area in the manuscript. Similarly, in lines 260-269, after stating the mutation rate we proceed onto suggesting possible means of improving it. Again, we believe this current structure is a best-fit and cannot find a more appropriate place to move this information. We are open to ideas though in case the reviewer has a specific location in mind.

Line 280 – 282

The authors should include more information to justify their choice of methods and clearly demonstrate how reliable the knockdown is. I presume the authors selected the RH2B sgRNA target 1 clutch 3 embryos because they see high somatic activity (4/13, 30.8%). Is this the case?

From the 4 embryos subjected to PCR screening for the RH2B gene, those PCR amplicons were subcloned and how many clones were sequenced? Figure 3A would suggest maybe 16 clones were sequenced. Is this also the case for tyr?

According to the text, authors subcloned and sequenced tyr PCR amplicons from embryos that came from clutch 9 (line 279). However, Figure 3 described subcloned sequenced from clutch 8 (lines 316, 331).”

- More information on our reasons for choosing the clutches for subcloning has now been provided (lines 246-247). They were indeed chosen as clutches with a high amount of somatic activity. The numbers (‘X/X’) after each sequence in Figure 3 reflects the number of the particular variant sequences detected out of the total sampled subcloned colonies per individual (i.e., M1 = 6, M2 = 9, M3 = 10, and M4 = 7 for RH2B; M1 = 10, M2 = 7, M3 = 10, M4 = 8 for tyr). This has now been more clearly stated in the caption for Figure 3. For RH2B-M1 and -M4, and tyr-M2 and -M4, we have also now performed NGS amplicon sequencing that yielded a few previously undetected variants (see lines 341-348 and sequences in Figure 3 A, B). We have changed the text to reflect our use of tyr clutch 12 in our subcloning analysis.

Note: what was previously clutch 8 is now clutch 12 due to since adding the RH2B Exon 1 injected clutches (no. 8 to 11).

In regard to line 308, if the data positions the authors to do so, they can write that “no wild-type sequences were detected in any of the sequenced clones (n=# of sequenced clones).” However, lines 203-204 and 281 suggest the authors may be observing compound heterozygous fish.

- We have changed this to read that no wild-type sequences were detected in any of the sequenced clones for RH2B.

Line 292 – 294. A graphical presentation can be made for the indels generated. In a bar graph, a column may show how many in frame mutations and frameshift mutations were made that were deletions and another column of the insertions that were in frame or frameshift mutations.

Another type of graph to depict the distribution of CRISPR/Cas9 induced mutations is to show number of mutations on the y axis and the base pair changes (- and +) plotted on the x axis. In this graph, authors will have bars that indicate deletion range and bars that indicate insertion sizes.

- We have now added two column graphs (see Figures 3 C and 4 C) showing the number of frameshift and in-frame mutations detected in each individual embryo. Although we have not separated this by insertions and deletions, as this does not add any information to the figure, where the nature and size (+/- Δbp) of the mutations are already stated after each variant sequence.

Figure 2

In Figure 2C and Figure 2D, labels for which fragment is uncut and which are cleaved can be presented. The in vitro experiment is successful, but another control is to run the sgRNA alone. In Figure 2C, the large overexposed bands may be tyr1 and tyr2 sgRNAs and not cut DNA.”

- We have now added boxes around cut fragments. Both target genes have negative controls (either no sgRNA and Cas9), and positive controls (sgRNA and no Cas9 for RH2B, or Cas9 and no sgRNA for tyr). This shows that fragment size differences were due to CRISPR/Cas9 cutting activity. We have since amended the figure to depict the correct positive control for tyr. The lower smeared region very possibly contains sgRNA; however, its persistence in the positive control for tyr (containing no sgRNA) raises doubt and it may simply be remnant primer contained in the PCR product (no clean-up routine was done). In any case, the well-defined fragment (now boxed in the figure) above the smear region cannot be sgRNA, as this fragment exceeds 100bp and our sgRNAs are only 23bp in length.

References:

1. Salis P, Lorin T, Lewis V, Rey C, Marcionetti A, Escande ML, Roux N, Besseau L, Salamin N, Sémon M, Parichy D, Volff JN, Laudet V. Developmental and comparative transcriptomic identification of iridophore contribution to white barring in clownfish. Pigment Cell Melanoma Res. 2019 May;32(3):391-402. doi: 10.1111/pcmr.12766. Epub 2019 Jan 29. PMID: 30633441; PMCID: PMC6483885.”

Reviewer #3

Reviewer #3: The manuscript by Mitchell LJ et al. describes CRISPR-Cas 9 genome editing in anemonefish. The authors efficiently demonstrate successful editing of two separate target sites- RH2B and Tyrosinase producing gene at G0 of A.ocellaris embryos. The experiments are well designed, performed with appropriate controls and the manuscript is well written. The manuscript will be potentially important to study gene functions in other reef fishes.

“1. Can authors comment on how is the CRISPR-Cas 9 protocol described here is novel and different from previously published CRISPR-Cas 9 editing done in other fishes?”

- Please refer to our detailed answer to reviewer 1.

2. Can this protocol be applied to precisely delete specific nucleotides at the target site and also Knock-in specific genes?

- Certainly, but this would involve heavily modifying the sgRNA design to perform knock-ins. Such an approach is already used with some success in other organisms [e.g., for a review on zebrafish see Albadri et al. (2017). Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish]. For an in-frame knock in this commonly requires homologous recombination (rather than non-homologous end joining for knockouts) with the insertion of DNA flanked by homologous sequences or arms on either side of the region of interest. Similarly, to delete specific nucleotides this could be enabled by microhomology mediated end-joining (MMEJ) by using short homologous arms flanking the target sequence. We have added brief mention in our discussion (lines 443-451) for how both these approaches could be introduced to perform other edits using anemonefish.

3. The authors show loss of function phenotype in tyr KO embryos, is there a functional phenotype to demonstrate loss of R2BH in the embryos? Probably by Immunohistochemical analysis of opsin trafficking in photoreceptors?

- We have not performed any functional characterisation for RH2B mutants, as we believe this is beyond the methodologically focused aspect of the paper and more suitable for an extensive experimental paper that will examine in detail the function of different opsins in anemonefish colour vision. Certainly, in-situ hybridisation and/or immunohistochemistry will be part of that future study, as will behavioural testing of colour vision.

4. Comments on any off-target/unpredicted mutations.

- Our methods section (lines 172-182) describes our off-target analysis when designing our sgRNA sequences. This used an inbuilt off-target screen in the ‘Find CRISPR sites’ function of the software ‘Geneious’. The application offers a number of candidate sgRNA sequences at a predefined region of interest for the user to select. As part of the metrics given for each sgRNA there is a specificity score calculated using a renowned method developed by the Zhang Lab at MIT. Please also refer to our detailed answer to Reviewer 1 on this question.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

1 Dec 2021

CRISPR/Cas9-mediated generation of biallelic G0 anemonefish (Amphiprion ocellaris) mutants

PONE-D-21-10870R1

Dear Dr. Mitchell,

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Reviewer #2: Reviewer Comments to Author(s):

The revised manuscript describes the use of CRISPR/Cas9 for genome editing in reef fish or anemonefish. While CRISPR/Cas9 mediated mutagenesis has been performed in a previous publication (1), the rational is that no protocol has detailed how to inject anemonefish to generate mosaic mutants or complete knockouts in the F0 generation. In the revised manuscript (PONE-D-21-10870_R1), the authors established a system for implementing CRISPR/Cas9 to produce genome edited reef fish. Although the authors microinjection technique affected the survivability of injected embryos, the overall success rate of CRISPR/Cas9 mediated mutagenesis was measurable. Heritability of CRISPR/Cas9 induced mutations were not detected in the injected reef fish due to the model systems long generation time constraints. However, the authors generate mosaic founder (F0) embryos that closely resemble true null mutations, at least in the case of tyr. The ability to generate F0 mutants will allow testing the functional importance of genes, although off-target effect is more common with this approach. The research presented provides the groundwork for CRISPR/Cas9 application in anemonefish and has a greater impact on the advancement of the use of non-traditional model organisms in scientific discovery.

Overall, the authors have addressed all comments, questions and concerns in the revised manuscript. The scientific arguments and interpretation are accurate and consistent with the results presented in the revised manuscript. The revised manuscript readability and flow of the text is presented with sufficient information for readers to follow the rationale, procedures and glean insights into the application of the CRISPR/Cas9 system for use on reef fish and other model organisms with longer generation times.

References:

1. Salis P, Lorin T, Lewis V, Rey C, Marcionetti A, Escande ML, Roux N, Besseau L, Salamin N, Sémon M, Parichy D, Volff JN, Laudet V. Developmental and comparative transcriptomic identification of iridophore contribution to white barring in clownfish. Pigment Cell Melanoma Res. 2019 May;32(3):391-402. doi: 10.1111/pcmr.12766. Epub 2019 Jan 29. PMID: 30633441; PMCID: PMC6483885.

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6 Dec 2021

PONE-D-21-10870R1

CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants

Dear Dr. Mitchell:

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If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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.

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