Genotyping MUltiplexed-Sequencing of CRISPR-Localized Editing (GMUSCLE): An Experimental and Computational Approach for Analyzing CRISPR-Edited Cells

CRISPR J. 2023 Oct;6(5):462-472. doi: 10.1089/crispr.2023.0021.


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-α/β 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.

MeSH terms

  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Cas Systems* / genetics
  • Gene Editing* / methods
  • Genome
  • Genotype


  • CRISPR-Associated Protein 9