GMUSCLE

Genotyping MUltiplexed-Sequencing of CRISPR-Localized Editing

CRISPR-Cas9 gene editing is widely used to create genetic modified cells. The genetic editing results in cellular heterogeneity, allelic diversity, and randomness of de-novo sequences, which constitute a major challenge in genotyping CRISPR-Cas9-edited cells.

We introduce a streamlined and cost-effective protocol along with the computational tool GMUSCLE, to sequence and genotype the products of CRISPR-Cas9 gene editing with great depth, high accuracy, high efficiency, and handy utilization.

GMUSCLE enables the quantitative and qualitative identification of the genotypes of CRISPR-Cas9-edited cells from sequencing data.

Reference: Zhang P., et al. Genotyping MUltiplexed-Sequencing of CRISPR-Localized Editing (GMUSCLE): An experimental and computational approach for analyzing CRISPR-edited cells. The CRISPR Journal. 2023. [PMID:37824834] [Paper]

Human Reference Genome:
GRCh37
GRCh38

Chromosome: [chr1-22, chrX, chrY]
Start Position: [integer, > 0]
End Position: [integer, > 0]
Read Count Cutoff: [integer, >= 0]
Genotype Proportion Cutoff: [decimal, 0 ~ 1]
Upload Data:

Single Sample
Upload a demultiplexed .fastq.gz file for one sample. [example]
Targeted genomic region of the example is chr21:34715869-34716164 (GRCh37).

Read Type:
Single-end

Pair-end
R1:
R2:

Multiple Samples
Upload a .zip file containing demultiplexed .fastq.gz files for multiple samples. [example]
Include a text file listing the sample names (one per line, without .fastq.gz). [example]
If pair-end, the fastq must be named as '_R1.fastq.gz' and '_R2.fastq.gz'.
Targeted genomic region of the example is chr21:34715869-34716164 (GRCh37).

Read Type: Single-end Pair-end
Sample FASTQ:
Sample Names:

Note:
File format - upload data in .fastq.gz for faster transfer and processing.
File size limit - uploads must be < 50 MB. For larger datasets, please use the standalone version.
Genotype filtering - genotypes < 1% will not be plotted, but will appear in downloaded result.
Indel display - indels outside the given genomic region will not be shown in the alignment.

GMUSCLE

GMUSCLE

Genotyping MUltiplexed-Sequencing of CRISPR-Localized Editing

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Developer| Peng ZHANG Ph.D.

Lab Head| Jean-Laurent CASANOVA M.D., Ph.D.

Laboratory| Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University

Last Update| June 30, 2025

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