Establishment of Rice Base Editing Technology Based on HyperCas12a and DddA
At present,the single-strand DNA deaminase and CRISPR/Cas9-mediated plant base editing system can effectively achieve specific base replacement on the genome and have been widely used in crop defect gene correction and endogenous gene directed evolu-tion.Additionally,Cas12a,which recognizes TTTV PAM(protospacer adjacent motif)can be used as a complement to Cas9,which recognizes NGG PAM.CRISPR/Cas12a-mediated base editors had been reported in plants,but all of them were constructed by fusing single-stranded DNA deaminase with the CRISPR/Cas12a system.This study tried to optimize the CRISPR/Cas12a-mediated rice base editing system by introducing double-stranded DNA deaminase into it,based on a large number of pre-laboratory studies.Firstly,the highly efficient Cas12a variant hyperCas12a was combined with the highly efficient single-stranded DNA deaminase(TadA9 or hAID∗Δ)to construct base editing tools rBE92 and rBE94.Then,the non-toxic mutant of double-stranded DNA deaminase DddA was introduced into rBE92 and rBE94 to construct base editors rBE92e/f and rBE94e/f.And the editing activity of various base editing tools was eval-uated in detail using the stable genetic transformation system of rice mediated by Agrobacterium tumefaciens.By rBE92 and rBE94,which were constructed by hyperCas12a and high-efficiency single-strand DNA deaminase(TadA9 or hAID∗Δ),the expected base editing events were not detected at the target site in the A.tumefaciens-mediated stable genetic transformation test of rice;furthermore,the new editing tools rBE92e/f and rBE94e/f were upgraded by introducing DddA-GSVG,and their editing efficiency was detected in rice,where the base editing events of C-to-T/G or A-to-G were detected in the two targets of Pita and OsCPK13 which induced by rBE92f and rBE94e respectively.The editing efficiency was 4.16%in Pita and 1.04%in OsMPK13,and the editing window was from the 10th to the 16th position at the 3' end of the PAM sequence.Based on the findings,it is believed that the hyperCas12a-DddA-mediated base editing technology for rice genome have successfully established.This study provides another new direction for optimizing and enriching rice base editing tools,which helps to achieve base editing and directed evolution of regions with rich T bases such as promoters or terminators in rice.
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