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Updated: Oct 5, 2025
11:35
Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
12.8K
Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity.
Rongjie Fu1, Wei He1, Jinzhuang Dou1
1Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, 78957, USA.
Nature Communications
|January 26, 2022
Summary
New rules for CRISPR/Cas9 genome editing specificity were discovered, improving prediction of off-target effects and enabling precise allele-specific editing for cancer mutations.
Area of Science:
- Molecular Biology
- Genetics
- Bioinformatics
Background:
- CRISPR/Cas9 genome editing relies on guide RNA (gRNA) and DNA sequence complementarity.
- Understanding sequence-dependent rules for off-target effects is crucial for precise genome editing.
Purpose of the Study:
- To systematically investigate sequence determinants of CRISPR/Cas9 off-target effects.
- To develop a predictive model for Cas9-mediated off-target activity.
- To explore allele-specific genome editing strategies.
Main Methods:
- Developed a dual-target system to measure relative cleavage rates (off-on ratios) for 1902 gRNAs across 13,314 synthetic targets.
- Analyzed sequence rules governing off-targeting, including guide-intrinsic mismatch tolerance (GMT) and combinatorial mismatch effects.
- Developed the MOFF (Model Of guide-RNA-guided Fidelity) predictor.
Main Results:
- Identified two key sequence rules for off-targeting: GMT and an "epistasis-like" combinatorial effect of mismatches.
- The combinatorial effect is linked to R-loop formation free energy and explained by a multi-state kinetic model.
- MOFF accurately predicts Cas9 off-target effects.
- Demonstrated improved allele-specific editing selectivity using mismatched guides and MOFF predictions on 18 cancer hotspot mutations.
Conclusions:
- Sequence rules governing CRISPR/Cas9 specificity have been elucidated.
- MOFF provides a powerful tool for predicting and minimizing off-target effects.
- The combinatorial mismatch effect enables enhanced allele-specific genome editing strategies for therapeutic applications.
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