Yuen Garmen, Khan Fehad J, Gao Shaojian, Stommel Jayne M, Batchelor Eric, Wu Xiaolin, Luo Ji
Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
Undergraduate Scholarship Program, National Institutes of Health, Bethesda, MD, USA.
Nucleic Acids Res. 2017 Nov 16;45(20):12039-12053. doi: 10.1093/nar/gkx843.
CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform.
CRISPR/Cas9是一种用于基因敲除研究和功能基因组筛选的强大基因编辑工具。CRISPR的成功实施通常需要Cas9在一群细胞中引发高效的靶点敲除。在本研究中，我们调查了几个关键因素在确定CRISPR敲除效率中的作用，这些因素包括靶点拷贝数的变化、sgRNA引导序列的内在效力以及Cas9和sgRNA的表达水平。使用具有不同EGFP转基因拷贝数的同基因克隆细胞系，我们发现CRISPR敲除对靶点拷贝数相对不敏感，但高度依赖于sgRNA引导序列的效力。动力学分析表明，大多数靶点突变发生在Cas9/sgRNA转导后的5至10天之间，而具有不同效力的sgRNA在敲除时间进程和终末期敲除效率方面存在差异。我们表明，Cas9和sgRNA的长时间低水平表达通常无法引发靶点突变，特别是当sgRNA的效力也很低时。我们的研究结果为CRISPR/Cas9在哺乳动物细胞中的行为提供了新的见解，可用于该平台未来的改进。
