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Updated: Mar 29, 2026

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Published on: November 1, 2024
PAM multiplicity marks genomic target sites as inhibitory to CRISPR-Cas9 editing
Abba Malina1, Christopher J F Cameron2, Francis Robert1
1Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada.
Abstract:
In CRISPR-Cas9 genome editing, the underlying principles for selecting guide RNA (gRNA) sequences that would ensure for efficient target site modification remain poorly understood. Here we show that target sites harbouring multiple protospacer adjacent motifs (PAMs) are refractory to Cas9-mediated repair in situ. Thus we refine which substrates should be avoided in gRNA design, implicating PAM density as a novel sequence-specific feature that inhibits in vivo Cas9-driven DNA modification.
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