Single nucleotide polymorphism in the genomic target affects the recombination efficiency of CRISPR/Cas9-mediated gene editing in zebrafish

Abstract

The CRISPR/Cas9 gene editing tool is becoming increasingly popular in the scientific community as one of the most efficient gene editing tools because of its high specificity and precision in targeted mutagenesis. A typical CRISPR system includes two components: a single guide RNA (sgRNA or gRNA) and a CRISPR associated endonuclease (Cas9). In CRISPR, the specificity of the Cas9 protein in cleaving the target DNA is decided by the homology of the spacer sequence, particularly the seed sequence (8–10 bases at the 3′ end of the spacer sequence) and any mismatch in the seed sequence abolishes the target cleavage and the subsequent generation of indels. Here, using Poly (A)specific ribonuclease (PARN) as an example in zebrafish, we show that the presence of single nucleotide polymorphism (SNP) in the sgRNA target, particularly in the seed sequence, affects the Cas9 activity resulting in a reduced recombination efficiency of CRISPR. Thus, while selecting target sites, prior screening for SNPs in the chosen targets across different wild-type lines/strains could be one of the easiest and effective ways of avoiding reduced recombination efficiency of CRISPR-mediated gene editing in zebrafish.