Establishment And Optimization Of CRISPR/Cas12a-Mediated Genome Editing And Targted Gene Replacement Systems In Rice Plants

Rice are one of the major crops which play a crucial role in food security globally.Following population growth,global warming,decreased agricultural acreage and water shortages,production of rice in China still remains challenging.Improvements in plant breeding are essential to feed a growing population under a warming and deteriorative global environment.CRISPR/Cas-mediated genome editing technologies enable sequence-specific precise modifications of DNA sequences including targeted mutagenesis for gene knock-out,single base substitution and gene/allele replacement in vivo,and thus has the potential to create a novel,sustainable agriculture.Genome editing has become a top priority and a commanding height in international strategic competition in global biological seed industry.Given that the most valuable alleles in food crops are conferred by differences of one or several single nucleotide polymorphisms?SNPs?,or defined insertion/deletions?indels?of a gene fragment in either promoter region or a gene's encoding region,introduction of these elite alleles into commercialized cultivars without deleterious gene drags through genome editing-mediated homology-directed repair?HDR?will be time-saving and greatly facilitate the breeding process.However,up to date,only a few successful reports have been documented in plants.Conducting HDR has been difficult,especially in plants,for two main reasons:1)the frequency of HDR is very low;2)limited DRT delivered into plant cells for HDR due to the cell wall.HDR efficiency has become the technical bottleneck of CRISPR/Cas mediated crop gene replacement,research hotspot and competitive focus in this field.Cas12a?previously named as Cpf1?,a new class 2 type V CRISPR/Cas system,is guided by a single CRISPR RNA?cr RNA?,which makes it easier for multiplex gene editing.However,the target sequences for genome editing byCRISPR/Cas12a are restricted to 5'-TTTV-3'PAM,thus limiting the availability of suitable target sitesand reducing the practical utility of Cas12a in plants.Moreover,it was reported that Cas12a produced along 5'protruding end which may facilitate the pairing and insertion of repair templates and thus HDR.However,Cas12a-mediated HDR has not been documented yet.To overcome 5'-TTTV-3'PAM restriction and achieve targeted gene replacement in plants by CRISPR/Cas12a-mediated HDR,we did several aspects of researches and the results are as following.1)Expanding the scope of CRISPR/Cas12a-mediated genome editing in rice:To test theendogenous genome editing capability of LbCas12a G532R/K595R variant LbCas12a?RR?andG532R/K538V/Y542R variant LbCas12a?RVR?in rice,we selected OsPDS and OsSBEIIb as thetarget genes.To reduce the disturbance of T-rice PAM on genome editing efficiency,we separatedthe crRNAs with HH ribozyme and HDV ribozyme units.Then,we did multiplex gene editing byusing two loci in one gene or simultaneously targeting two genes.Our results demonstrated thatLbCas12a?RR?enabled multiplex editing of target genes containing 5'-TYCV-3'PAM in rice at ahighest editing efficiency of 51%.Genome editing at this non-canonical PAM site expanded thescope of LbCas12a-mediated genome editing in rice.2)Synthesis-dependent repair of Cpf1-induced double-strand DNA breaks enables targeted genereplacement in rice:Cpf1-crRNA produces double strand DNA breaks?DSBs?with long 5'protruding ends,which may facilitate the pairing and insertion of repair templates throughhomology-directed repair?HDR?for targeted gene replacement and introduction of the desired DNAelements at specific gene loci for crop improvement.However,the potential mechanism underlyingHDR of DSBs generated by Cpf1-crRNA remains to be investigated and the inherent low efficiencyof HDR and poor availability of exogenous donor DNA as repair templates strongly impede the useof HDR for precise genome editing in crop plants.Here,we provide evidence of synthesis-dependentrepair of Cpf1-induced DSBs,which enables us to precisely replace the wild-type ALS gene with theintended mutant version that carries two discrete point mutations conferring rice plants herbicideresistance.Our observation that the donor repair template?DRT?with only left homologous arm issufficient for precise targeted allele replacement offers a better understanding of the mechanismunderlying HDR in plants,and greatly simplifies the design of DRTs for precision genome editingin crop improvement.Moreover,by using the codon-optimized Cas12a and driving two Ribozyme-crRNA-Ribozyme unit by two different pol III promoters respectively,we generated 15 homozygousALS edited rice plants.Our results provided fundamental technical basis for CRISPR/Cas12a-mediated gene replacement in crop improvement.3)Precise gene replacement in rice by RNA transcript-templated homologous recombination:RNA transcript-templated HDR has been reported in yeast and human cell lines,but not yet ingenome editing and plants.To test the feasibility of RNA transcript as donor template for DNA repairand for targeted gene replacement through genome editing,an RNP complex comprising of LbCpf1-crRNA and RNA transcripts was assembled in vitro and transformed into rice calli by bombardment,we first confirmed RNA transcript-templated homologous recombination in rice calli by allelic-specific PCR amplification and sequencing.Furthermore,we used droplet digital PCR?ddPCR?todetect HDR events and to evaluate the RNP-mediated HDR efficiency when the repair templateswere RNA transcripts,DNA,or both DNA and RNA,respectively.And then,we constructed thevectors by placing two RCR?HH ribozyme-cr RNA-HDV ribozyme?units and an RDR?HHribozyme-donor repair template-HDV ribozyme?unit or target 1-donor-target 2 in a tandem mannerunder the control of the OsU3 promoter and terminated by a NOS terminator.Upon transcription,both the mature crRNAs and the repair RNA template were released simultaneously.We thentransformed these vectors into rice calli through bombarment,and successfully recovered transgene-free herbicide resistant lines with precisely modified rice ALS gene following segregation in the T₁generation.Collectively,we proved for the first time that RNA transcript could serve as a templatefor DNA repair for targeted gene replacement in rice through CRISPR/Cas12a-mediated genomeediting.In summary,we expanded the editing scope of CRISPR/Cas12a in rice by using the LbCas12a?RR?variant.Furthermore,we established CRISPR/Cas12a-mediated gene replacement in rice using DNA repair template containing two homology arms or only the left homology arms,and dissected the potential mechanism of HDR.Finally,we successfully established a CRISPR/Cas12a mediated gene replacement system in rice by using RNA transcript as repair template.In a word,our researches provided a fundamental basis and technical support for creating elite novel crop germplasm and breeding for improved varieties by genome editing to ensure global food security.