等位基因特异性DNA甲基化是位于差异甲基化区域(DMRs)的最重要的印记标记,和异常基因组印迹DNA甲基化与一些人类疾病有关,包括Prader-Willi综合征和癌症.因此,精确编辑等位基因特异性甲基化基因的有效策略的开发对于阐明印迹元件的功能和纠正人类疾病中的印迹障碍至关重要。为了发现基于CRISPR/Cas系统的可行的等位基因特异性基因组编辑工具,这是一种在各种生物中有效的基因靶向技术,我们检测了金黄色葡萄球菌Cas9(SaCas9)和化脓性链球菌Cas9(SpCas9)对DNA甲基化干扰的靶向效率.我们发现,使用dCas9-Tet1催化结构域(CD)的靶向DNA去甲基化增强了SaCas9而不是SpCas9的靶向效率,但使用Dnmt3l-Dnmt3a-dCas9被靶向DNA甲基化抑制。体外裂解测定进一步证明,在合成的含CpG的环境中,SaCas9核酸酶活性被5-甲基胞嘧啶(5mC)抑制。用ChIP-Q-PCR的进一步分析表明,SaCas9的非甲基化序列靶向依赖于SaCas9对非甲基化序列的结合偏好。利用SaCas9的这一特点,我们成功获得了两个印迹基因的非甲基化等位基因偏向性靶向胚胎/小鼠,H19和Snrpn,效率相对较高,分别为28.6%和47.4%,分别。这些结果表明SaCas9的靶向效率由于DNA甲基化而大大降低。通过使用SaCas9,我们通过优先靶向非甲基化基因座,成功地实现了印迹基因的等位基因特异性基因组编辑。
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.