Negative DNA supercoiling induces genome-wide Cas9 off-target activity.
Mol Cell
; 83(19): 3533-3545.e5, 2023 Oct 05.
Article
in En
| MEDLINE
| ID: mdl-37802026
ABSTRACT
CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
CRISPR-Cas Systems
/
Gene Editing
Language:
En
Journal:
Mol Cell
Journal subject:
BIOLOGIA MOLECULAR
Year:
2023
Document type:
Article
Affiliation country:
Reino Unido