Harnessing CRISPR-Cas9 fidelity for therapeutic genome editing: from off-target mechanisms to delivery design

CRISPR-Cas9, an RNA-guided immune system adapted for genome editing, enables sequence-specific DNA cleavage through a programmable guide RNA (gRNA).This programmability has expanded therapeutic genome editing across diverse disease contexts, and the first CRISPR-Cas9-based therapy (Casgevy) has now been approved.However, Cas9 tolerates partial mismatches between the gRNA and the genomic target, and the resulting off-target cleavage can introduce mutations, large deletions, and chromosomal rearrangements.Off-target effects further depend on the dose, persistence, and tissue distribution of the active Cas9-gRNA complex.Over the past decade, sensitive detection methods, along with Cas9 and gRNA engineering, have substantially improved editing precision, supporting safer therapeutic translation.In this review, we discuss the mechanism of off-target cleavage and the strategies developed to mitigate it, including high-fidelity Cas9 variants, chemically and structurally modified gRNAs, and delivery formats.