Veeranagouda Yaligara, Debono-Lagneaux Delphine, Fournet Hamida, Thill Gilbert, Didier Michel
Molecular Biology and Genomics, Translational Sciences, Sanofi R&D, Chilly-Mazarin, France.
Curr Protoc Mol Biol. 2018 Jan 16;121:31.14.1-31.14.11. doi: 10.1002/cpmb.53.
The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells). Here we described a next-generation sequencing method called CRISPR-Cas9 Edited Site Sequencing (CRES-Seq) for the efficient and high-throughput screening of CRISPR-Cas9-edited clones. CRES-Seq facilitates the precise genotyping up to 96 CRISPR-Cas9-edited sites (CRES) in a single MiniSeq (Illumina) run with an approximate sequencing cost of $6/clone. CRES-Seq is particularly useful when multiple genes are simultaneously targeted by CRISPR-Cas9, and also for screening of clones generated from multiallelic genes/polyploid cells. © 2018 by John Wiley & Sons, Inc.
成簇规律间隔短回文重复序列-Cas9（CRISPR-Cas9）基因编辑系统的出现，使得在真核细胞中能够低成本、短时间且高效地创建特定突变体。由于CRISPR-Cas9系统通常会在靶位点产生一系列突变，因此一个成功的基因编辑项目需要仔细选择编辑后的克隆。这个过程可能非常具有挑战性，尤其是在处理多等位基因和/或多倍体细胞（如癌细胞和植物细胞）时。在此，我们描述了一种名为CRISPR-Cas9编辑位点测序（CRES-Seq）的新一代测序方法，用于高效、高通量筛选CRISPR-Cas9编辑后的克隆。CRES-Seq能够在单次MiniSeq（Illumina）运行中对多达96个CRISPR-Cas9编辑位点（CRES）进行精确基因分型，每个克隆的测序成本约为6美元。当多个基因同时被CRISPR-Cas9靶向时，CRES-Seq特别有用，也可用于筛选多等位基因/多倍体细胞产生的克隆。© 2018约翰威立父子公司版权所有
