Development And Optimization Of CRISPR Systems For Rice Genome Editing

CRISPR-Cas9,the third generation of gene editing technology,has been widely rolled out in the many fields such as basic research,disease prevention and germplasm innovation.Under this situation,no matter from the perspective of mechanism research and practical application,exploration and development of the third generation of gene editing technique in rice,one of the major food crops in the world and one kind of important endogen model plants,are significant.However,the function of CRISPR-Cas9 in rice depends on several specific nucleotides in genome,to wit,PAM region,which greatly limits the edting scope of CRISPR-Cas9 system in rice genome.In order to break the shackle that SpCas9 employs NGG as PAM in rice,this study setting out from the application of Cas9 variants,development of Cas9 editing potential with non-canonical PAMs,establishment the CRISPR-Cpf1 system and optimization of CRISPR editing systems in rice,established CRISPR-VQR system in rice for recognizing NGA PAM,CRISPR-VRER system in rice for recognizing NGCG PAM and CRISPR-Cpf1 system in rice for recognizing T-rich PAM by codon optimization and site-specific mutagenesis,which remarkably enriched the rice gene editing toolbox.The result showed all three editing systems were able to introduce mutations effectively.Thus,by analyzing,the editing range within rice genome was expaned to more than 2-fold that of original Cas9.As to off-target effect,VQR and VRER performed similarly as original Cas9,while no off-target event of Cpf1 was detected during this test.In addition,this study,on one hand,optimized current CRISPR-Cas9-VQR system in rice with significantly improving in systems editing efficiency,which was up from 3-fold to 7-fold,by employing Actin1 promoters and modifying sgRNA.On other hand,for CRISPR-Cpf1,we explored crRNA and its way to express,which suggested,comparing to single crRNA array,matured crRNAs expressed by tRNA-crRNA system performed better at inefficient target sites.These results show further optimizing is achieved,which provides convenience and reference for using these systems in rice even in other species.Besides,this study found wild Cas9 protein could efficiently recognize and edit around NAG PAM in rice for the first time.According to the test,in stable transformations,the Cas9's efficiency of recognizing NAG PAM was as same as that of recognizing NGG PAM,and NGG PAM and NAG PAM could be employed at same test for multiple editing,which roughly doubled the flexibility in applying regular CRISPR-Cas9 technology.Moreover,the results of off-target effect showed Cas9 produced less off-target effect when recognizing NAG PAM than NGG PAM,which could reduce potential off-target effect in practical application.