Chen Baohui, Hu Jeffrey, Almeida Ricardo, Liu Harrison, Balakrishnan Sanjeev, Covill-Cooke Christian, Lim Wendell A, Huang Bo
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA.
Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA.
Nucleic Acids Res. 2016 May 5;44(8):e75. doi: 10.1093/nar/gkv1533. Epub 2016 Jan 5.
In order to elucidate the functional organization of the genome, it is vital to directly visualize the interactions between genomic elements in living cells. For this purpose, we engineered the Cas9 protein from Staphylococcus aureus (SaCas9) for the imaging of endogenous genomic loci, which showed a similar robustness and efficiency as previously reported for Streptococcus pyogenes Cas9 (SpCas9). Imaging readouts allowed us to characterize the DNA-binding activity of SaCas9 and to optimize its sgRNA scaffold. Combining SaCas9 and SpCas9, we demonstrated two-color CRISPR imaging with the capability to resolve genomic loci spaced by <300 kb. Combinatorial color-mixing further enabled us to code multiple genomic elements in the same cell. Our results highlight the potential of combining SpCas9 and SaCas9 for multiplexed CRISPR-Cas9 applications, such as imaging and genome engineering.
为了阐明基因组的功能组织，直接可视化活细胞中基因组元件之间的相互作用至关重要。为此，我们对金黄色葡萄球菌的Cas9蛋白（SaCas9）进行了工程改造，用于对内源基因组位点进行成像，其显示出与先前报道的化脓性链球菌Cas9（SpCas9）相似的稳健性和效率。成像读数使我们能够表征SaCas9的DNA结合活性并优化其sgRNA支架。结合SaCas9和SpCas9，我们展示了双色CRISPR成像，能够解析间距小于300 kb的基因组位点。组合颜色混合进一步使我们能够在同一细胞中对多个基因组元件进行编码。我们的结果突出了将SpCas9和SaCas9结合用于多重CRISPR-Cas9应用（如成像和基因组工程）的潜力。
