Creation And Identification Of CircRNA Knockout Mutants In Rice

Circular RNA（circRNA）is a newly endogenous nocoding RNA（ncRNA）,it’s structure as closed loop,which is different from the traditional linear RNA structure.In order to connect 3’and 5’cohesive ends,circRNA’s exons or introns form a complete ring structure.This kind of structure won’t affect by RNA exonuclease,thus it’s more stable and conserved than linear RNA,and can exist in many types and large amount in organisms.CircRNA plays an important regulatory role in miRNA function research and gene transcription.Because of its unique molecular structure and potential genomic function,it has attracted widespread attention.Now,some circRNA have been found in animals and plants,but the function of circRNA which can be explained is very few,one reason of this condition is lack of circRNA mutants.In this study,we based on the genome oriented modification technology,obtained circRNA knockout mutant materials of five rice genome as Os02circ25329,Os06circ02797,Os03circ00204,Os01circ14411 and Os05circ02465.According to the mutant material which obtained,the homozygous mutant material is selected and sequenced,and the CRISPR-Cas9 modification efficiency of circRNA large segments knocking out in rice genome is analyzed.As for the selected mutant plants,later research will move on to transcriptome sequencing in order to analyze its function.The contents and results of this study are as follows:1.Aiming for Os02circ25329,Os06circ02797,Os03circ00204,Os01circ14411,Os05circ02465,we constructed five vectors based on CRISPR-Cas9,named pZJP053,pZJP054,pZJP055,pZJP056,pZJP057 respectively.Each vector contains two sgRNA sites.2.Rice genetic transformation is carried out by Agrobacterium tumefaciens,and274 transgenic rice plants were obtained,of which pZJP053 obtained 43 plants,pZJP054 obtained 15 plants,pZJP055 obtained 111 plants,pZJP056 obtained 61 plants,and pZJP057 obtained 44 plants.3.We detected the mutation of single locus in some transgenic positive plants.Among them,the mutation efficiency of pZJP053-sgRNA1 modified site is 56.5%;the mutation efficiency of pZJP053-sgRNA2 modified site is 47.8%.The mutation efficiency of pZJP054-sgRNA1 modified site is 60.0%,and the mutation efficiency of pZJP054-sgRNA2 site is 30.0%.Mutation efficiency of pZJP055-sgRNA1 modified site is 30.4%,and the mutation efficiency of pZJP055-sgRNA2 modified site is 8.7%.The mutation efficiency of pZJP056-sgRNA1 modified site is 100.0%,and the mutation efficiency of pZJP056-sgRNA2 modified site is 95.7%.The mutation efficiency of pZJP057-sgRNA1modified site is 47.8%,and the mutation efficiency of pZJP057-sgRNA2 modified site is47.8%.4.We detected the transgenic positive plants,and verified that we have obtained 23large fragments deletion T₀ mutant which targeted circRNA.Among them,pZJP053obtained 6 knockout mutants with a mutation efficiency of 14%.And pZJP054 obtained1 knockout mutants with a mutation efficiency of 6.7%.And pZJP055 obtained 5knockout mutants with a mutation efficiency of 4.5%.And pZJP056 obtained 5 knockout mutants with a mutation efficiency of 8.2%.And pZJP057 obtained 6 knockout mutants with a mutation efficiency of 13.6%.5.We selecte T₁ mutant plants in order to obtain the homozygous plants,in which circRNA Os02circ25329,Os03circ00204,and Os05circ02465 are successfully generated homozygous knock out mutated plants which contains no vector.This lays the foundation for the subsequent research of the functions analysis on circRNA.