Screening of High-Efficient sgRNA for Targeted Knockout of GhAGL16 Gene in Cotton
[Objective]As an important negative transcriptional regulator in cotton MADS-box gene family,AGL16 plays an important role in resisting drought and salt stress.Virus-induced gene editing(VIGE)was used to screen sgRNAs that knockout the cotton GhAGL16 and verify the specificity of these sgRNAs,which laid a foundation for the creation of cotton agl16 mutants.[Method]Three sgRNAs could knockout GhAGL16 were predicted based on the actual GhAGL16 genomic sequence cloned on subgroup A and D in cotton YZ-1;Three CLCrV-AtU6-26::GhAGL16-sgRNAs vectors were constructed based on the cotton leaf crumple virus(CLCrV)-mediated VIGE system;The expression of Cas9 in Cas9 over-expression(Cas9-OE)plants was detected by qPCR to determine whether Cas9 was stably genetically expressed;Three CLCrV-AtU6-26::GhAGL16-sgRNAs vectors were transformed respectively into Cas9-OE cotton cotyledons and detected the mutations of the three targets by PCR/RE;The secondary structures of three GhAGL16-sgRNAs were analyzed by bioinformatics;Hi-TOM high-throughput sequencing was performed on mutant plants to determine the efficiency of gene editing.Meanwhile,the off-target rate of GhAGL16-sgRNA2 mutant plants were identified to detect the specificity of gene editing.[Result]Three sgRNAs capable of simultaneously knocking out GhAGL16-A and D subgroups were successfully constructed.The detection results of Cas9 expression showed that Cas9 was stably expressed in different Cas9-OE cotton plants.PCR/RE mutation detection results showed that GhAGL16-sgRNA2 could be effectively used for the knockout of GhAGL16.Different mutation types with base deletions appeared at the target sites of cotton subgroups A and D,while GhAGL16-sgRNA1 and GhAGL16-sgRNA3 were two invalid sgRNAs.The secondary structure analysis results of three GhAGL16-sgRNAs indicated that GhAGL16-sgRNA1 and GhAGL16-sgRNA3 might have a phenomenon that the guide sequence was easy to pair with other sequences and difficult to unwind,which interfered with the recognition of the target site by the guide sequences and lead to the invalid sgRNA.To further quantify the editing efficiency of GhAGL16-sgRNA2 on GhAGL16,the mutation detection results of each Cas9-OE plant transformed with CLCrV-AtU6-26::GhAGL16-sgRNA2 showed that six of the nine Cas9-OE plants were mutated,with a mutation efficiency of 66.67%.In addition,Hi-TOM high-throughput sequencing results showed that the editing efficiency of GhAGL16-sgRNA2 for GhAGL16 was 13.69%-54.42%.The off-target identification results showed that no off-target phenomenon was detected at the four predicted off-target sites,indicating that GhAGL16-sgRNA2 not only has high gene editing efficiency,but also has specific gene editing specificity.[Conclusion]A sgRNA that can effectively knocking out the GhAGL16 was obtained by transforming Cas9-OE cotton using the CLCrV-mediated VIGE system,providing an ideal sgRNA for creating cotton agl16 mutants.
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