Optimizing plant adenine base editor systems by modifying the transgene selection system.

Dear Editor,

Classical CRISPR‐Cas systems introduce a DNA double‐strand break (DSB) at target genomic loci. In plant, DSBs are typically repaired through the error‐prone nonhomologous end joining (NHEJ) pathway and result in small InDels (Chen et al., 2019). Recently, base editors (BEs), including cytosine BEs (CBEs) and adenine BEs (ABEs), were developed to introduce precise nucleotide substitutions by combining the CRISPR‐Cas system with engineered nucleotide deaminases (Gaudelli et al., 2017; Komor et al., 2016). These BEs enable precise conversions between A∙T and G∙C pairs in the eukaryotic genome without introducing DSBs. ABE was developed by fusing directly evolved E. coli TadA tRNA adenosine deaminase (ecTadA*7.10) to SpCas9 nickase (Gaudelli et al., 2017). In plants, the efficiency of ABE tools is generally limited and varies greatly among different targets (Hua et al., 2019). Several efforts have been made to enhance the A∙T‐to‐G∙C conversion activity of ABE by adjusting the number and location of nuclear location signals (NLSs) or the architecture of ABE (Hua et al., 2019; Li et al., 2018). However, the efficiency improvement of ABE is still eagerly wanted for robust base conversions in plant genome. Here, we report selection‐based enrichment strategies to achieve highly efficient adenine editing in rice.

We previously established a plant ABE tool (the pHUN411‐ABE vector) exhibiting limited efficiency (Li et al., 2019). Another study indicated that by using an ACC‐1 single guide RNA (sgRNA), the ABE system can introduce a T‐to‐C conversion corresponding to the dominant C2186R mutation of OsACC, which confers aryloxyphenoxypropionate (APP) herbicide resistance in rice (Li et al., 2018). This process may provide a marker to select base‐edited plants under herbicide pressure thus may enhance editing efficiency by enriching cells with a functional ABE system. To test this hypothesis, the ACC‐1 sgRNA and the sgRNA for desired target were constructed into single pHUN411‐ABE vector for simultaneous expression (Figure 1a). The vectors were introduced into rice (Oryza sativa cv. Nipponbare) calli via Agrobacterium‐mediated transformation. The resistant calli were selected by hygromycin with or without the APP herbicide haloxyfop‐R‐methyl for 14 days. For each transformant, ~200 newly emerged calli were collected as a sample to analyse editing by amplicon‐based next generation sequencing (NGS, BioProject PRJNA588580). The frequency of base conversion in T9 of the Pid3‐1 site and T5/T6 of the WX‐2 site was increased under double selections relative to that selected by hygromycin alone (Figure 1b). However, no significant enhancement in the frequency of nucleotide substitution was obtained at A6 of the WX‐1 site. Plants were regenerated under continuous selections, and then mutations were identified. Up to 31.3% of the hygromycin‐selected plants carried base conversion at the desired targets, whereas the mutant frequencies were greater than 50.0% under double selection of hygromycin and the herbicide (Figure 1c). The results demonstrate that the selection of ACC‐1‐edited plants might enhance base editing of the ABE system at some targets. Because the activity of ABEs may vary greatly among different targets (Hua et al., 2018; Kang et al., 2018; Li et al., 2019; Yan et al., 2018), the presence of a functional ABE for ACC‐1 editing cannot guarantee the editing of the coexpressed sgRNA in the same cells. In addition, the coedited herbicide selection marker needs to be removed through segregation by self‐crossing or backcrossing in plants, which may limit the practical application of this strategy.

Figure 1

Optimizing plant ABE for base editing in the rice genome. (a) sgRNA expression structure for ABE‐mediated herbicide selection. The target sequences were indicated in bottom. The PAM was underlined. (b) Editing efficiencies at the targets with or without herbicide selection. The transformed calli were selected by 50 mg/L hygromycin alone (Hyg) or in combination with 2 μm herbicide (Hyg + Her). The frequency of base conversions in the editing window was calculated by counting reads of edited mutants compared to total clean reads with three biological replicates. *, P < 0.05; **, P < 0.01, t‐test. (c) The edited mutants generated by coexpressed sgRNAs. d, Schematic representation of the STTU ABEs. (e) Base editing efficiency of the STTU ABE systems. (f) The edited mutants generated by the STTU ABE systems. (g) ABE expression in the hygromycin‐selected plants of the STTU systems. Total RNA was extracted from regenerated ABE‐WX‐1 plants. A specific primer on Cas9 region was used to determine the expression level of ABE. ∆∆Ct was showed. n = 12. (h) The selection and rescue of later‐generation plants transformed with pPUN411‐ABEH vector. The T0 transgenic seeds were germinated at 28 °C in a 16 h/8 h light cycle. Selections on seeds of a representative pPUN411‐ABEH‐WX‐1line were shown.

BE expression level is closely correlated with editing efficiency (Koblan et al., 2018). Thus, base conversions should be more easily produced in plants with higher expression levels of BE. To obtain regenerated plants with high ABE expression levels, single transcriptional and translational unit (STTU) ABE systems were constructed by fusing hygromycin phosphotransferase (HPT) to the N or C terminus of the ecTadA‐ecTadA*7.10‐nSpCas9 region with a self‐cleavage 2A peptide (Figure 1d). The ABE coding region in pHUN411‐ABE was first replaced by HPT‐ABE (HABE) or ABE‐HPT (ABEH) fusion. To avoid duplicate HPTs, the original HPT in the vector backbone was replaced by a Mannose‐6‐phosphate isomerase (PMI) marker, generating the pPUN411‐HABE and pPUN411‐ABEH vectors (Figure 1d). The constructs were introduced into rice by Agrobacterium‐mediated transformation and selected by hygromycin. In resistant calli after 14‐day‐selection, amplicon‐based NGS indicated HABE generated significantly higher editing frequencies at 4 out of 6 desired sites compared with ABE (Figure 1e, BioProject PRJNA576084). At T9 of the Pid3‐1 site, the frequency of the conversion generated by HABE was increased as much as 2.7‐fold that achieved by ABE. Compared with the editing efficiencies of ABE, those of ABEH were significantly increased by 1.9‐ to 4.5‐fold at all 6 sites. The results indicate that the editing activity of ABE could be enhanced by HPT fusion. Interestingly, the editing efficiencies of ABEH were significantly higher than those of HABE at 4 sites, suggesting that C‐terminal HPT fusion may enhance the editing efficiency of the plant ABE system to a great extent than N‐terminal fusion. The base editing induced by HABE and ABEH was further determined in the regenerated plants (Figure 1f). Unlike the limited mutants generated by the unmodified ABE, the majority of transgenic plants of the HPT‐fused ABEs carried base conversions in the editing window of the target. Using the ABEH tool, >97.9% plants were edited at all targets, which suggests that the pPUN411‐ABEH vector can provide robust and efficient base editing in rice. During stable transformation, the cells with stronger expression of the selection marker will grow faster under selection pressure and have a greater opportunity to regenerate plants. However, in standard ABE systems, such as the pHUN411‐ABE system, ABE and HPT are typically expressed in different cassettes. Therefore, antibiotic‐resistant cells with high levels of HPT may not have enough expression of ABEs. In this report, we provide a strategy to fuse ABE and HPT in a STTU system, which can be expected to result in synchronization of their expression levels. Antibiotic selection on HPT expression thus would enrich cells with high ABE expression level. To test this hypothesis, the transcript levels of the different ABEs in the regenerated plants were determined by quantitative reverse transcription PCR (qRT‐PCR). The ABE expression levels of the pPUN411‐HABE/ABEH plants were significantly higher than those of the pHUN411‐ABE plants (Figure 1g, one‐way ANOVA), confirming the stronger expression of ABE in the STTU system. Although ABE expression did not significantly differ between the ABEH and HABE plants, we believe that the C‐terminal HPT of the ABEH fusion may avoid the incomplete transcription or translation of ABE in the resistant cells, thereby providing increased editing activity in hygromycin‐resistant cells.

CRISPR genome editing systems are frequently introduced into plants by Agrobacterium‐mediated stable transformation. The inserted T‐DNA fragment may need to remove in edited lines by segregation. Most of plant CRISPR systems use negative selection, such as an antibiotic or herbicide selection. Generally, negative selection is lethal to untransformed cells. If negative selection is applied to segregate the T‐DNA‐free lines in the T1 generation, desirable plants without T‐DNA insertions may be subject to toxicity and thus hardly to be recovered. In contrast, positive selection methods (e.g. mannose) typically inhibit but do not kill untransformed cells. To screen T‐DNA‐free progeny lines, the T0 seeds of pPUN411‐ABEH were selected by hygromycin or mannose pressure for 4 days, and then, sensitive seeds were rescued without selection for another 4 days (Figure 1h). We found that the recovery of mannose‐selected seeds was much easier than that of seeds selected by hygromycin. Furthermore, PCRs with sequence‐specific primers confirmed that 97.8% (45 out 46) rescued mannose‐sensitive plants were T‐DNA‐free. These results suggested the PMI‐mannose selection in genome editing system can facilitate the segregation of T‐DNA‐free plants. Taken together, our results provide an efficient and easy‐to‐use ABE system for plant adenine base editing. More importantly, the study offers a general strategy to optimize efficiency of stable transformation‐based plant genome editing.

Conflict of interest

The authors declare no conflict of interest.

Author contributions

P.W. designed the experiments and wrote the manuscript. P.W., J.Y. and X. Z. supervised the project. J.L., R.Q., Y.Z., S.X. and X.L. performed all the experiments. J.L., R.Q. and Y.Z. analysed the results.

Figure S1 PCR identification of T‐DNA region in the plants rescued from the mannose‐sensitive seeds.

Table S1 Selection and rescue of pPUN411‐ABEH seeds under hygromycin or mannose.

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