High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis.
High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis.
复制标题
DOI：
10.1038/srep15587
复制
发表时间：
2015-10-26
影响因子：
4.6
通讯作者：
Li S
中科院分区：
文献类型：
--
作者：
Ramlee MK;Yan T;Cheung AM;Chuah CT;Li S
序列特异性合成核酸酶工程的最新进展为基因表达的遗传操作提供了巨大的机会，以便在体内研究它们的细胞功能。然而，由于固有的限制和高成本，目前检测靶向人类细胞中这些可编程核酸酶诱导的插入/缺失（indel）突变的基因分型方法与敲除克隆的高通量筛选不相容。在这里，我们描述了一种以高通量方式对克隆CRISPR/Cas9介导的突变体进行基因分型的有效方法，包括使用直接裂解缓冲液直接从培养的细胞中提取粗基因组DNA，以及荧光PCR与毛细管凝胶电泳偶联。该技术还允许在单个克隆中对多重基因靶向进行基因分型。总的来说，这种节省时间和成本的技术能够规避当前基因分型方法的局限性，并支持核酸酶诱导突变体的高通量筛选。
Recent advances in the engineering of sequence-specific synthetic nucleases provide enormous opportunities for genetic manipulation of gene expression in order to study their cellular function in vivo. However, current genotyping methods to detect these programmable nuclease-induced insertion/deletion (indel) mutations in targeted human cells are not compatible for high-throughput screening of knockout clones due to inherent limitations and high cost. Here, we describe an efficient method of genotyping clonal CRISPR/Cas9-mediated mutants in a high-throughput manner involving the use of a direct lysis buffer to extract crude genomic DNA straight from cells in culture, and fluorescent PCR coupled with capillary gel electrophoresis. This technique also allows for genotyping of multiplexed gene targeting in a single clone. Overall, this time- and cost-saving technique is able to circumvent the limitations of current genotyping methods and support high-throughput screening of nuclease-induced mutants.