Allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci using Staphylococcus aureus Cas9 (SaCas9)

Allele-specific DNA methylation is the most important imprinting marker localized to differentially methylated regions (DMRs), and aberrant genomic imprinted DNA methylation is associated with some human diseases, including Prader-Willi syndrome and cancer. Thus, the development of an effective strategy for the precise editing of allele-specific methylated genes is essential for the functional clarification of imprinting elements and the correction of imprinting disorders in human diseases. To discover a feasible allele-specific genome editing tool based on the CRISPR/Cas system, which is an efficient gene-targeting technique in various organisms, we examined the targeting efficiency of Staphylococcus aureus Cas9 (SaCas9) and Streptococcus pyogenes Cas9 (SpCas9) in response to DNA methylation interference. We found that the targeting efficiency of SaCas9, but not SpCas9, was enhanced by targeted DNA demethylation using the dCas9-Tet1 catalytic domain (CD) but suppressed by targeted DNA methylation using Dnmt3l-Dnmt3a-dCas9. An in vitro cleavage assay further demonstrated that SaCas9 nuclease activity was inhibited by 5-methylcytosine (5mC) in a synthesized CpG-containing context. Further analysis with ChIP-Q-PCR demonstrated that the non-methylated sequence targeting of SaCas9 depends on the binding preference of SaCas9 to non-methylated sequences. Taking advantage of this feature of SaCas9, we have successfully obtained non-methylated allele-biased targeted embryos/mice for two imprinting genes, H19 and Snrpn, with relatively high efficiencies of 28.6% and 47.4%, respectively. These results indicate that the targeting efficiency of SaCas9 was strongly reduced by DNA methylation. By using SaCas9, we successfully achieved allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci.