Related Experiment Video
Updated: Apr 21, 2026
CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions
Published on: November 1, 2024
Off-target assessment of CRISPR-Cas9 guiding RNAs in human iPS and mouse ES cells
E-Pien Tan1, Yilong Li, Martin Del Castillo Velasco-Herrera
1Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.
Abstract:
The CRISPR-Cas9 system consists of a site-specific, targetable DNA nuclease that holds great potential in gene editing and genome-wide screening applications. To apply the CRISPR-Cas9 system to these assays successfully, the rate at which Cas9 induces DNA breaks at undesired loci must be understood. We characterized the rate of Cas9 off-target activity in typical Cas9 experiments in two human and one mouse cell lines. We analyzed the Cas9 cutting activity of 12 gRNAs in both their targeted sites and ∼90 predicted off-target sites per gRNA. In a Cas9-based knockout experiment, gRNAs induced detectable Cas9 cutting activity in all on-target sites and in only a few off-target sites genome-wide in human 293FT, human-induced pluripotent stem (hiPS) cells, and mouse embryonic stem (ES) cells. Both the cutting rates and DNA repair patterns were highly correlated between the two human cell lines in both on-target and off-target sites. In clonal Cas9 cutting analysis in mouse ES cells, biallelic Cas9 cutting was observed with low off-target activity. Our results show that off-target activity of Cas9 is low and predictable by the degree of sequence identity between the gRNA and a potential off-target site. Off-target Cas9 activity can be minimized by selecting gRNAs with few off-target sites of near complementarity.
Insights
CRISPR-Cas9 gene editing shows low off-target DNA breaks in human and mouse cells. Careful guide RNA selection minimizes unintended Cas9 cutting, ensuring predictable and safe genome editing applications.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas9 is a powerful gene editing tool with broad applications.
- Understanding Cas9's off-target DNA cutting is crucial for successful gene editing and screening.
- Off-target activity must be quantified to ensure the safety and efficacy of CRISPR-Cas9 applications.
Purpose of the Study:
- To characterize the rate of Cas9 off-target activity in typical experimental conditions.
- To analyze Cas9 cutting efficiency at targeted and predicted off-target sites across different cell lines.
- To determine the predictability of Cas9 off-target effects based on sequence complementarity.
Main Methods:
- Evaluated Cas9 cutting activity of 12 guide RNAs (gRNAs) in human 293FT, human-induced pluripotent stem (hiPS), and mouse embryonic stem (ES) cells.
- Analyzed both on-target and approximately 90 predicted off-target sites per gRNA.
- Assessed cutting rates and DNA repair patterns, including clonal analysis in mouse ES cells.
Main Results:
- Cas9 cutting was detected at all on-target sites and only a few off-target sites genome-wide.
- Cutting rates and DNA repair patterns showed high correlation between the two human cell lines.
- Biallelic Cas9 cutting was observed in mouse ES cells with low off-target activity.
Conclusions:
- Cas9 off-target activity is generally low and predictable based on sequence identity between the gRNA and potential off-target sites.
- Off-target activity can be minimized by selecting gRNAs with limited near-complementary off-target sites.
- These findings support the reliable application of CRISPR-Cas9 in gene editing and screening.
