Precise Modifications of Both Exogenous and Endogenous Genes in Rice by Prime Editing

Abstract

Harnessing genetic diversity and the introduction of elite alleles from wild relatives or landraces into commercial cultivars has been a major goal in crop breeding programs. Precise modification of the plant genome through clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) (CRISPR/Cas)-mediated homology-directed repair (HDR) offers a great promise to introduce elite alleles from wild relatives or landraces into commercialized cultivars in the short term. Various strategies have been attempted for precise targeted gene/allele replacement or gene insertion and tagging through CRISPR/Cas systems in plants (Baltes et al., 2014Baltes N.J. Gil-Humanes J. Cermak T. Atkins P.A. Voytas D.F. DNA replicons for plant genome engineering.Plant Cell. 2014; 26: 151-163Crossref PubMed Scopus (279) Google Scholar, Gil-Humanes et al., 2016Gil-Humanes J. Wang Y. Liang Z. Shan Q. Ozuna Serafini C. Sánchez-León S. Baltes N. Starker C. Barro F. 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However, due to the intrinsic lower frequency of HDR in plant cells, insufficient availability of a donor repair template, and the non-homologous end-joining (NHEJ) is the predominant DNA repair pathway in plants (Baltes et al., 2014Baltes N.J. Gil-Humanes J. Cermak T. Atkins P.A. Voytas D.F. DNA replicons for plant genome engineering.Plant Cell. 2014; 26: 151-163Crossref PubMed Scopus (279) Google Scholar, Li et al., 2019Li S. Li J. He Y. Xu M. Zhang J. Du W. Zhao Y. Xia L. Precise gene replacement in rice by RNA transcript-templated homologous recombination.Nat. Biotechnol. 2019; 37: 445-450Crossref PubMed Scopus (50) Google Scholar), it remains challenging, especially in crop plants. Thus, it is essential to further exploit more efficient precision genome-editing technology in order to accelerate crop improvement. Recently, a prime editing system, which enables targeted insertions, deletions, and all 12 classes of point mutations, without requiring double-strand breaks or a DNA donor repair template, was shown to function efficiently in mammalian cells (Anzalone et al., 2019Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (911) Google Scholar). In this report, a third generation of prime editor (PE3) was engineered by fusing a mutated M-MLV-RT (Moloney murine leukemia virus reverse transcriptase) to the C terminus of a catalytically impaired Cas9 (H840A) (Cas9 nickase, nCas9), and programmed with a prime editing guide RNA (pegRNA) composed of a single chimeric guide RNA (sgRNA) targeting the specific site, a primer-binding site (PBS), and a reverse transcription (RT) template encoding the desired edit. The PE3 complex binds the target DNA and nicks at the non-target strand, and uses a nicked genomic DNA strand as a primer for the synthesis of an edited DNA flap by extension using the RT template on the pegRNA. Subsequent DNA repair incorporates the edited DNA flap on the non-target strand and further copies the edit into the complementary target strand, resulting in stably edited DNA. At the same time, another nicking sgRNA at various distances from the nicks induced by pegRNA was used to direct a second cut on the target strand to increase the chances of repairing this strand to match the edited sequence (Figure 1A). The prime editing system could substantially expand the scope and capabilities of CRISPR/Cas9 in precise modification of plant genomes for base transition/transversion and targeted gene/allele replacement in crop improvement. It remains unclear whether a similar strategy will work efficiently in plant species, especially in crops. In this study, to evaluate the feasibility and efficacy of PE3 prime editor in precision genome editing in rice, we first mutated Cas9 into nCas9(H840A) in our pCXUN-Ubi-NLS-Cas9-NLS-PolyA-E9 vector (Supplemental Figure 1A), in which the expression of the rice codon optimized Cas9 with a nuclear localization signal (NLS) at both the N terminus and C terminus was driven by a maize ubiquitin promoter (ubi) and terminated by a PolyA sequence and a pea (Pisum sativum) Rubisco small subunit E9 terminator. We then fused the rice codon optimized M-MLV-RT sequence through a 99-bp linker at the C terminus of nCas9 (H840A) and a 42-bp linker before the C-terminal NLS to generate pCXUN-Ubi-NLS-nCas9(H840A)-Linker1(33aa)-M-MLV-RT-Linker2(14aa)-NLS-PolyA-E9 (Supplemental Figure 1B). A cassette containing the Actin promoter and the Nos terminator was cloned into the above vector, yielding a basic prime editor vector pCXUN-Ubi-NLS-nCas9(H840A)-Linker1(33aa)-M-MLV-RT-Linker2(14aa)-NLS-PolyA-E9-Actin-Nos (hereafter referred to as the prime editor-basic) (Supplemental Figure 1C). In order to test the feasibility of the prime editor-basic vector in precision editing of exogenous gene, we further mutated the hptII gene in our prime editor-basic vector at position Gly 45 (GGA) to TGA and Tyr 46 (TAT) to TAG to generate a prime editor-basic-hptII-mutant vector (Supplemental Figure 1D). The introduction of these two point mutations will disable the ability of the hptII gene to confer hygromycin resistance on rice calli during selection. We then designed a pegRNA composed of an sgRNA, a 28-bp RT (including two synonymous mutations and two mutations to restore the two stop codons into original GGA and TAT) and a 13-bp PBS, which are reversely complementary to the non-target strand, and another nicking sgRNA for a second cut, which is located at a distance of 50-bp upstream from the nick induced by pegRNA on the non-target strand (Figure 1B). Then, taking advantage of the automatic tRNA self-processing activity in vivo (Xie et al., 2015Xie K. Minkenberg B. Yang Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system.Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 3570-3575Crossref PubMed Scopus (591) Google Scholar), we used the polycistronic tRNA strategy to simultaneously produce pegRNA and nicking sgRNA. We cloned the tRNA-pegRNA-tRNA-sgRNA-tRNA-PolyA complex into this vector to generate prime editor-hptII mutant vector, in which the pegRNA and nicking sgRNA were separated by two tRNAs and driven by a single constitutive rice Actin promoter and terminated by a PolyA sequence to increase the stability of pegRNA and nicking sgRNA transcripts and a Nos terminator (Supplemental Figure 1E). We delivered this vector into rice (Japonica cv. Zhonghua 11) calli by particle bombardment. Subsequently, the calli were treated at 30°C for 4 h and then subjected to induction and selection on medium containing 50 mg/L hygromycin for 2–3 weeks, then transferred to medium containing 75 mg/L hygromycin for another 2–3 weeks. Compared with a negative control, which was transformed with the prime editor-basic-hptII-mutant vector, we observed vigorous differentiation and growth of some calli on the induction medium after 1 week and 4 weeks of selection, respectively (Figure 1B). We then transferred these vigorously grown calli onto regeneration medium for 3–4 weeks to generate stable lines. Among 148 calli bombarded, we obtained 32 hygromycin resistance calli. Treating the plants regenerated from each original bombarded callus as a single event, the genotype of each event was determined by Sanger sequencing of the PCR amplicons using the primer set listed in Supplemental Table 1, followed by decoding. We recovered three independent events with a precisely modified hptII gene. Among 129 plants detected, we achieved 15 individual homozygous plants with precise edits (representative lines #5 and #15) (Figure 1B). The precision editing efficiency was 9.38% (3 of 32). The co-existence of both precise edited hygromycin resistance plants with the non-edited lines in one cluster may help these lines to survive after two rounds of hygromycin selection. We also detected a chimera line (line #8) containing one allele with precisely edited hptII, and another two with a 43-bp insertion either at the nicking sgRNA target site or the pegRNA target site. This 43-bp insertion is homologous to the fragment located between the nicking sgRNA and pegRNA, indicating the random insertion of this fragment by an NHEJ pathway. We also targeted a rice endogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (OsEPSPS) for prime editing. Introduction of a triple amino acid substitution (T169I, A170V, and P173S) (TIAVPS) in OsEPSPS may confer a higher level of glyphosate resistance on rice (Perotti et al., 2019Perotti V.E. Larran A.S. Palmieri V.E. Martinatto A.K. Alvarez C.E. Tuesca D. Permingeat H.R. A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate-resistant Amaranthus hybridus population from Argentina.Pest Manag. Sci. 2019; 75: 1242-1251Crossref PubMed Scopus (28) Google Scholar). We designed a pegRNA composed of an sgRNA, a 59-bp RT (including three synonymous mutations and triple TIAVPS mutations), and a 13-bp PBS, which are reversely complementary to the non-target strand, and another nicking sgRNA for the second cut, which is located at a distance of 66-bp downstream from the nick induced by pegRNA on the non-target strand (Figure 1C). Following a similar strategy to that of hptII prime editing, we shuttled the pegRNA and nicking sgRNA assembly in a structure of tRNA-pegRNA-tRNA-sgRNA-tRNA-PolyA into our prime editor-basic vector to generate primer-editor-EPSPS vector (Supplemental Figure 1F). We delivered this vector into rice calli by particle bombardment following the same transformation and tissue culture procedure as that of hptII prime editing. Among 73 calli bombarded, we obtained 45 independent transgenic plants. The genotypes of these plants were determined by Sanger sequencing of the PCR amplicons using the primer set listed in Supplemental Table 1, followed by decoding. Among these plants, we achieved one heterozygous line (line #12) with one allele that had the desired TIAVPS precise edits, whereas another had a 10-bp insertion between pegRNA and sgRNA target sites (Figure 1C). The precision editing efficiency was 2.22% (1 of 45). We also recovered another three partial edited lines (Figure 1C). One line had partial edits (line #2), while another two had additional 17-bp and 40-bp insertions at the sgRNA target site, respectively (lines #9 and #20). The different efficiencies observed in prime editing of hptII and OsEPSPS might be due to the different RT length, location of the second cut, as well as the intrinsic nature of target genes as observed in previous a report on mammalian cells (Anzalone et al., 2019Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (911) Google Scholar). To evaluate the specificity of the prime editor in rice, we examined the off-target possibility for each on-target site. No off-target effects were found at the potential off-target sites (CRISPR-GE, http://skl.scau.edu.cn/) in the tested lines (Supplemental Table 2). We then genotyped the T0 lines with precise edits by PCR using the DNA extracted from three independent leaves from each line. Except for the chimeric lines, all sampled leaves from individual plants carried homogenous mutations. Taken together, we have established an efficient prime editing system in rice and successfully achieved homozygous and heterozygous stable lines with the desired edits in both exogenous and endogenous genes, providing a feasible and effective tool for precision editing of the rice genome. As an effective and universal strategy, the plant prime editing system established in this study can also be applied in other crop species, providing the useful tool for improving crops in a user-defined manner. L.X. conceived the project. H.L., J.L., J.C., and L.Y. performed the experiments. L.X. wrote the manuscript. This work is partly funded by the Ministry of Agriculture and Rural Affairs of China ( 2019ZX08010001-001-007 and 2019ZX08010003-002-003 ), and the Central Non-Profit Fundamental Research Funding supported by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences ( S2018QY05 ) to L.X. No conflict of interest declared. Download .pdf (.38 MB) Help with pdf files Document S1. Supplemental Materials and Methods, Supplemental Figure 1, Supplemental Tables 1 and 2, and Supplemental References