| CRISPR-Cas is a revolutionary technology of life science,which plays an important role in the basic research of plant and crop breeding.Off-target effect are bottlenecks of CRISPR-Cas.To minimize the Off-target effect in plants,three of Sp Cas9 variants(evo Cas9,Sniper-Cas9,LZ3 Cas9),and es Cas9 and Super Fi-Cas9-1 which were synthesized by structure-guided mutagenesis variants,were verified of off-target effects in Arabidopsis.Firstly,we selected PDS3,FLS2,GL2,TT4,BRI1 and FER genes for targeted mutagenesis.Sp Cas9 and Sp Cas9-HF1 were used as off-target negative and low-off-target positive controls,respectively.T7E1 assay and deep sequencing results showed that all of variants induced effectively targeted mutations in protoplasts,and the editing patterns was as same as Sp Cas9.Then,the editing efficiency of LZ3 Cas9 is similar to Sp Cas9,but the editing efficiency of Sp Cas9-HF1,evo Cas9,Sniper-Cas9 and es Cas9 were all reduced.Subsequently,off-target editing was performed by using artificial Off-target guide RNA.The results showed that LZ3 Cas9 could reduce the off-target editing efficiency but did not affect the targeted editing efficiency with multiple Off-target guide RNA.Although offtarget editing could not be detected in plants,the off-target editing rates of Sp Cas9-HF1,Sniper-Cas9 and LZ3 Cas9 were all reduced at several off-target sites of Sp Cas9 in protoplasts.Considering the targeted editing efficiency,LZ3 Cas9 minimize Off-target effect and maintain editing efficiency,in comparison with Sp Cas9.Next,we mutated LZ3 Cas9 to LZ3 Sp RY for expanding targeting scope.The results showed that among the 16 designed PAM types,targeted mutations were induced in 14 PAM types,which is NGG,NGA,NGC,NAG,NAA,NAT,NAC,NTT,NTC,NTG,NCT,NCC,NCA and NCG.The results show that LZ3 Sp RY expand the targeting scope of LZ3Cas9.In addition,LZ3 Sp RY enhanced specificity comparing with Sp RY on the off-target sites.Furthermore,we created a novel variant,Super Fi-Cas9-1,to Minimize off-target effect in plants.Likely Sp Cas9,Super Fi-Cas9-1 show robust ability of targeted mutagenesis in protoplasts and plant,and then the Off-Target detection is ongoing. |
