Resrarch And Application Of Genome Editing Tool Of CRISPR-Cas12b/c2c1 In Plant

Withsimplicity,efficiency,flexibility and unexpensive,the CRISPR/Cassystem as the third generation of genome editing tool,has been proven to be effective for targeted gene editing in a wildly range of organisms and becomes the revolutionary tool in lifesciences.In addition to targeted insertion,deletion andsubstitution applications,CRISPR/Cassystem also achieves targeted gene activation andsuppression.Furthermore,CRISPR/Cassystem have been improved and expanded by new Cas potein.By these progresses,CRISPR technology is close to the dream tool for geneticmanipulation.Cas12b contains a conserved RuvC endonuclease domain and Nuc domain.Cas12b is a dual-RNA-guided DNA endonuclease,and both crRNA and tracrRNA are required for its double-stranded DNA cleavage activity.Cas12b recognizes a AT-rich protospacer adjacentmotif(PAM)sequence located at the 5'-end of a target DNAsequence andmakesstaggered cuts distal to the PAMsite with 5' overhangs.To evaluate whether the engineered CRISPR-Cas 12bsystem can be used as an alternative genome editing tool in plants,weselected Bacillussp.V3-13(Bv)and Bacillus hisashii(Bh)Cas12b variants for targetedmutagenesis in Arabidopsis protoplasts.Firstly,BvCasl2b and BhCas12b v4 were codon-optimized and were attached nuclear localizationsignals at both ends.second,weselectedseveral targetsites across four genomic loci(PDS3,FLS2,GL2 and TT4)in Arabidopsis as aspacer to designsgRNAs.spCas9 constructs targeting thesame loci were added for comparison.The target region was examined using the T7 endonuclease(T7E1)assay to test whethermutagenesis was induced,and then deepsequencing analyses for indel frequencies and patterns.In total,themutagenesis efficiency ofselectedsitesshowsstrong differences depending on the target,ranging from 0 to amaximum above 4.3%efficiency.A deletion of 5 to 13 base pairs was themost common editing pattern at targetsites.The CRISPR-Cas 12b system allowsmultiplex genome editing and large genomic deletions in the genome,and does not tolerate?3mismatches.Finally,we observed the loss-of-function phenotype as the nullmutant of target gene from transgenic plants.These results demonstrate that CRISPR-Cas 12b is a potential genome-editing tool for plant genome editing.