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Updated: Jun 8, 2025
08:23
CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Published on: November 1, 2024
531
Seed sequences mediate off-target activity in the CRISPR-interference system
Neha Rohatgi1, Jean-Philippe Fortin2, Ted Lau3
1Genentech Computational Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA; Roche Informatics, Hoffman-La Roche Canada, 7070 Mississauga Road, Mississauga, ON, Canada.
Cell Genomics
|November 7, 2024
Summary
The CRISPR interference (CRISPRi) system can cause widespread gene silencing off-target effects. These unintended impacts are mainly due to seed sequence complementarity between the sgRNA and genomic DNA.
Area of Science:
- Molecular Biology
- Genetics
- Functional Genomics
Background:
- CRISPR interference (CRISPRi) is a key technology for gene silencing in research.
- Systematic investigation of CRISPRi off-target activity is lacking.
Purpose of the Study:
- To investigate genome-wide off-target activity of the CRISPRi system.
- To understand the impact of off-target effects on gene expression.
Main Methods:
- Utilized a genome-wide CRISPRi-Cas9 single-guide RNA (sgRNA) library.
- Analyzed off-target activity and its effects on gene expression.
Main Results:
- Off-target effects in CRISPRi are pervasive, impacting gene expression directly and indirectly.
- Most off-targets are linked to the seed sequence (3' half of sgRNA spacer) complementarity with PAM-proximal genomic regions.
- Off-target binding stability is influenced by seed sequence length and mismatch tolerance.
Conclusions:
- CRISPRi off-target activity is a significant consideration in functional genomics.
- Understanding seed sequence interactions is crucial for predicting and mitigating CRISPRi off-target effects.
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