A simple method based on Sanger sequencing and MS Word wildcard searching to identify Cas9-induced frameshift mutations

作     者：Jie, Hui Li, Zhuoling Wang, Ping Zhao, Linjie Zhang, Qian Yao, Xiaomin Song, Xiangrong Zhao, Yinglan Yao, Shaohua 

作者机构：Sichuan Univ West China Hosp State Key Lab Biotherapy Canc Ctr 17 3rd Sect South Renmin Rd Chengdu 610041 Sichuan Peoples R China 

出 版 物：《LABORATORY INVESTIGATION》 (Lab. Investig.)

年 卷 期：2017年第97卷第12期

页      面：1500-1507页

核心收录：

学科分类：1001[医学-基础医学(可授医学、理学学位)] 10[医学] 

基　　金：Hi-Tech Research and Development (863) Program of China [2015AA020309] National Natural Science Foundation of China (NSFC) 

摘      要：Recent advances in targeted genome editing have enabled sequence-specific modifications in eukaryotic genomes. As it can be easily reprogrammed, the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 nuclease system has been studied extensively and is now a widely used genome editing tool. Generally, Cas9 nucleases are designed to target the coding regions in exons of protein-coding genes, which are expected to cause frameshift indel mutations and interrupt protein expression. In such cases, it is often necessary to separate single clones that harbor double frameshift mutant alleles from clones that harbor the wild-type allele or an in-frame mutant allele. We developed a simple and efficient method to identify frameshift mutations in diploid genomes based on Sanger sequencing and MS Word wildcard searching (SWS). As indel mutations induced by Cas9 are varied, Sanger sequencing of PCR products from a single mutant genome will generate double peaks that begin at the indel sites. By positioning the putative sequences deduced from the double peak regions in the sequencing graph onto the wild-type sequence by MS Word wildcard searching, it is possible to predict exactly how many nucleotides were deleted or inserted in each allele of the genome. The SWS strategy greatly facilitates the process of identifying single clones with biallelic frameshift mutations from pooled cells or model organisms.