For knockout of the chicken myostatin (MSTN) gene, the nickase target loci were designed in exon 1 of chicken MSTN gene (Figure 1A).
The Cas9-D10A nickase target sites were designed in exon 1 of chicken MSTN gene (Figure 1A).
In the MSTN knockout DF1 cells induced by the Cas9-D10A nickase, the various mutant genotypes of the mixed population were identified (Figure 2C).
In this study, we designed two adjacent (+7 bp offset, Figure 1B) but opposite single-strand breaks in chicken MSTN gene for Cas9-D10A nickase.
The red sequences are the targeted sites of the CRISPR-associated protein 9 (Cas9)-D10A nickase.
The data demonstrates strong activities versus monomeric wild-type Cas9 nucleases and nickases, as well as increased genomic specificities with no evidence of unwanted mutagenesis at off-target sites.
The specificity of fCas9 was shown to be at least four-fold higher than that of paired nickases at human genome loci with highly similar off-target sites elsewhere in the genome.