Optimization And Application Of Rice Base Editing Technology Based On CRISPR/SpRY System

Rice(Oryza sativa L.)is one of the most important crops in the world,supporting nearly half of the global population.However,rice cultivation is often affected by biotic as well as abiotic factors,which result in the failure of rice to grow properly and seriously affect the yield and quality of rice.Therefore,the development of novel rice varieties with desirable agronomic traits is necessary to maintain global food security.In recent years,gene editing technology has been developed to achieve precision breeding by precisely modifying the target gene or introducing a certain superior gene into the plant to produce superior agronomic traits.And base editing technology has become an effective and reliable tool for precision editing of plant genomes.Base editing technology achieve precise editing using adenine or cytosine base editors within the target range.At present,the commonly used base editor is based on Sp Cas9,which mainly recognizes NGG PAM,this greatly limits the editable range in plant genomes.Therefore,over the years,researchers have set out to develop a variety of highly efficient,and wide range of gene editing tools,so as to achieve efficient and accurate editing in the whole genome of plants,which will play an important role in promoting the study of plant genome function and crop breeding.In order to broaden the targeting scope of rice gene editing,in this study we performed rice codon optimization of two structurally engineered Sp Cas9 variants-SpG and-SpRY developed in animals and examined their PAM preferences as well as knockout and base editing efficiency.The results showed that SpG has preference for NG PAM,but its activity is not as good as Sp Cas9-NG previously reported by the team,and thus Sp Cas9-NG is more suitable for NG PAM editing in rice.In contrast,SpRY enables efficient editing at a wide range of genomic loci,exhibiting a preference for NGD and NAN PAM,in addition to recognizing NTC,NCT,NCC and NCG PAM loci,greatly extending the editing scope in rice.We fused SpRY(D10A)nickase with cytosine deaminase h AID*? to form cytosine base editor r BE66(h AID*?-SpRYn-UGI),and the results showed that could effectively recognize various atypical PAM to achieve the target nucleotide change of C>T,and r BE62(Tad A8e-SpRYn)could be formed by fusing SpRY(D10A)nickase with high activity adenine deaminase Tad A8 e to induce the target nucleotide change of A>G.The results showed that r BE62 was not only able to broaden the editing range in rice but also improved the efficiency of base editing.Furthermore,due to the broad recognition range of SpRY,this study revealed that it has high-frequency self-editing events,i.e.,editing of sgRNA in T-DNA,while the self-editing efficiency in the SpRY nickase-mediated base editor is very low.These results suggest that SpRY can be used for targeted editing of the rice genome,especially base editing,and greatly expands the scope of editing in plant genomes.