期刊
CRISPR JOURNAL
卷 6, 期 5, 页码 462-472出版社
MARY ANN LIEBERT, INC
DOI: 10.1089/crispr.2023.0021
关键词
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This study presents an experimental and computational method for genotyping CRISPR-Cas9 edited cells, including multiplexed sequencing and the software GMUSCLE. By sequencing CRISPR-Cas9 edited products and using GMUSCLE software for analysis, genotypes can be quantitatively and qualitatively identified, allowing for the selection and investigation of cell clones with specific genotypes.
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) creates double-stranded breaks, the repair of which generates indels around the target sites. These repairs can be mono-/multi-allelic, and the editing is often random and sometimes prolonged, resulting in considerable intercellular heterogeneity. The genotyping of CRISPR-Cas9-edited cells is challenging and the traditional genotyping methods are laborious. We introduce here a streamlined experimental and computational protocol for genotyping CRISPR-Cas9 genome-edited cells including cost-effective multiplexed sequencing and the software Genotyping MUltiplexed-Sequencing of CRISPR-Localized Editing (GMUSCLE). In this approach, CRISPR-Cas9-edited products are sequenced in great depth, then GMUSCLE quantitatively and qualitatively identifies the genotypes, which enable the selection and investigation of cell clones with genotypes of interest. We validate the protocol and software by performing CRISPR-Cas9-mediated disruption on interferon-alpha/beta receptor alpha, multiplexed sequencing, and identifying the genotypes simultaneously for 20 cell clones. Besides the multiplexed sequencing ability of this protocol, GMUSCLE is also applicable for the sequencing data from bulk cell populations. GMUSCLE is publicly available at our HGIDSOFT server and GitHub.
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