Targeted Genome Editing On Auxin Signaling Genes OsPINs And OsARFs Using CRISPR/Cas9 In Rice

CRISPR/Cas9 is a more powerful gene editing technology that can accurately edit the target gene.Since it’s first application 2013,it has been widely used in the study of gene function of many species in nature,including plants.Compared with other gene editing technologies,CRISPR/Cas9 is more accurate,efficient and simple,and cheap,therefore,it is more pratical for most of the laboratories.The birth of CRISPR/Cas9technology provides a new method and thingking for plant genetic engineering research.In order to study how auxin is transported from the cell to outside,and how does auxin participate in the regulation of plant growth and development,we used the CRISPR/Cas9 gene editing technology to knockout the OsPINs family genes and OsARF1,OsARF8 and OsARF10 in rice.CH vector（for OsPINs）and pBIN-sgRNA-Cas9-Osvector（for OsARFs）were constructed to generate corresponding CRISPR expression vectors.The resulting expression vectors were transformed into callus of rice by Agrobacterium-mediated transformation.We failed to obtain any mutants of OsPINs family genes using the CH vector.However,Based on pBIN-sgRNA-Cas9-Osvector,we successfully identified a heterozygous mutation of OsARF8,osarf8-1,from 28 transgenic plants screened by hygromycin resistance.The osarf8-1 lacked a C base at the 174^th bp of within the exon,resulting in change of leucine（Leu）to tryptophan（Trp）,causing frameshift of amino acid sequences and premature termination of the DBD domain of OsARF8.In addition,we also obtained 7 heterozygous lines of osarf10-1,which had with the deletions of three bases（AGG）from the 35^th to-37^th bp within the exon,resulting in the change of the 12^thglutamic acid（Glu）to aspartic acid（Asp）,and the 13^th glycine（Gly）to aspartic acid（ASP）.The knockout mutants of OsARF8 and OsARF10created by CRISPR/Cas9 in this study provide basis for further study on the function of OsARF8 and OsARF10 in rice.