Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
Bloomberg School of Public Health, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
Nat Commun. 2022 Dec 15;13(1):7776. doi: 10.1038/s41467-022-35476-y.
Direct visualization of point mutations in situ can be informative for studying genetic diseases and nuclear biology. We describe a direct hybridization genome imaging method with single-nucleotide sensitivity, single guide genome oligopaint via local denaturation fluorescence in situ hybridization (sgGOLDFISH), which leverages the high cleavage specificity of eSpCas9(1.1) variant combined with a rationally designed guide RNA to load a superhelicase and reveal probe binding sites through local denaturation. The guide RNA carries an intentionally introduced mismatch so that while wild-type target DNA sequence can be efficiently cleaved, a mutant sequence with an additional mismatch (e.g., caused by a point mutation) cannot be cleaved. Because sgGOLDFISH relies on genomic DNA being cleaved by Cas9 to reveal probe binding sites, the probes will only label the wild-type sequence but not the mutant sequence. Therefore, sgGOLDFISH has the sensitivity to differentiate the wild-type and mutant sequences differing by only a single base pair. Using sgGOLDFISH, we identify base-editor-modified and unmodified progeroid fibroblasts from a heterogeneous population, validate the identification through progerin immunofluorescence, and demonstrate accurate sub-nuclear localization of point mutations.
直接观察点突变的原位可视化对于研究遗传疾病和核生物学很有帮助。我们描述了一种直接杂交基因组成像方法，具有单核苷酸敏感性，通过局部变性荧光原位杂交（sgGOLDFISH）的单指导基因组寡探针，该方法利用 eSpCas9(1.1)变体的高切割特异性，结合经过合理设计的指导 RNA 加载超螺旋酶，并通过局部变性揭示探针结合位点。该指导 RNA 带有故意引入的错配，因此虽然可以有效地切割野生型靶 DNA 序列，但不能切割具有额外错配的突变序列（例如，由点突变引起的错配）。因为 sgGOLDFISH 依赖于 Cas9 切割基因组 DNA 来揭示探针结合位点，所以只有野生型序列而不是突变序列被探针标记。因此，sgGOLDFISH 具有区分仅相差一个碱基对的野生型和突变型序列的敏感性。使用 sgGOLDFISH，我们从异质群体中鉴定出碱基编辑器修饰和未修饰的早衰成纤维细胞，通过早衰蛋白免疫荧光验证鉴定结果，并证明了点突变的精确亚核定位。
