Creation of Novel Protein Variants with CRISPR/Cas9-Mediated Mutagenesis: Turning a Screening By-Product into a Discovery Tool.
Creation of Novel Protein Variants with CRISPR/Cas9-Mediated Mutagenesis: Turning a Screening By-Product into a Discovery Tool.
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用CRISPR/CAS9介导的诱变创建新型蛋白质变体：将筛选副产品变成发现工具。
DOI：
10.1371/journal.pone.0170445
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Doench JG
中科院分区：
文献类型：
--
作者：
Donovan KF;Hegde M;Sullender M;Vaimberg EW;Johannessen CM;Root DE;Doench JG
CRISPR/Cas9筛选已被证明是基因组学研究的通用工具。基于全基因组筛选的意外结果，我们开发了一种CRISPR/ cas9介导的诱变方法，利用易出错的非同源末端连接（NHEJ）产生的等位基因多样性来鉴定MAPK信号通路基因MEK1和BRAF的新的功能获得和耐药等位基因。我们定义了一种可扩展技术的参数，以轻松生成包含数千个内源性等位基因变体的细胞群，以绘制基因功能。此外，这些结果突出了一个意想不到但重要的现象，即Cas9诱导的功能获得等位基因是正常Cas9功能丧失筛选的固有副产品，应该在分析大规模阳性选择筛选的数据时进行研究。
CRISPR/Cas9 screening has proven to be a versatile tool for genomics research. Based on unexpected results from a genome-wide screen, we developed a CRISPR/Cas9-mediated approach to mutagenesis, exploiting the allelic diversity generated by error-prone non-homologous end-joining (NHEJ) to identify novel gain-of-function and drug resistant alleles of the MAPK signaling pathway genes MEK1 and BRAF. We define the parameters of a scalable technique to easily generate cell populations containing thousands of endogenous allelic variants to map gene functions. Further, these results highlight an unexpected but important phenomenon, that Cas9-induced gain-of-function alleles are an inherent by-product of normal Cas9 loss-of-function screens and should be investigated during analysis of data from large-scale positive selection screens.