Increasing The Efficiency Of CRISPR/Cas9-based Gene Editing By Suppressing RNAi In Plants

CRISPR/Cas9-based genome editing is a powerful and widely adopted technology for introducing specific mutations in animal and plant systems.Typically,a CRISPR/Cas9 system is composed of the endonuclease protein Cas9 and an engineered single-guide RNA(sgRNA)that specifically recognizes its target DNA sequence in the genome.In plants,stable Cas9 transformation and successful gene editing may be hindered by post-transcriptional transgene silencing,but the extent to which these mechanisms affect gene editing in plants has not been evaluated.Here we show that Arabidopsis mutants defective in gene silencing,such as rdr6 and dcl2 dcl3 dcl4,had greater Cas9 protein accumulation and increased gene editing efficiency.Based on this observation,we linked the CRISPR/Cas9 cassette to an artificial microRNA targeting RDR6(amiR-RDR6).The results show that RDR6 knockdown by amiR-RDR6 enhanced gene editing efficiency.Furthermore,the leaf phenotype caused by amiR-RDR6 served as a convenient visual marker for Cas9 screening.In summary,we demonstrated the hindering effect of RNAi in CRISPR/Cas9-based gene-editing and mitigated the negative impact of RNAi by repressing the expression of RDR6.This new strategy may therefore improve gene editing efficiency and enable convenient identification of Cas9-free mutants.