CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity

TLDR

CRISPR systems promise precise genome editing for research and therapy, but their target specificity remains largely unknown. Guide strands were designed with varying mismatches, and off‑target cleavage was assessed using T7 endonuclease I assays and Sanger sequencing. CRISPR/Cas9 targeting β‑globin and CCR5 caused substantial off‑target cleavage and chromosomal deletions, with repair generating diverse indels and point mutations, underscoring the need for careful design to avoid sites with mismatches near the PAM.

Abstract

The ability to precisely modify endogenous genes can significantly facilitate biological studies and disease treatment, and the clustered regularly interspaced short palindromic repeats (CRISPR) systems have the potential to be powerful tools for genome engineering. However, the target specificity of CRISPR systems is largely unknown. Here we demonstrate that CRISPR/Cas9 systems targeting the human hemoglobin β and C-C chemokine receptor type 5 genes have substantial off-target cleavage, especially within the hemoglobin δ and C-C chemokine receptor type 2 genes, respectively, causing gross chromosomal deletions. The guide strands of the CRISPR/Cas9 systems were designed to have a range of mismatches with the sequences of potential off-target sites. Off-target analysis was performed using the T7 endonuclease I mutation detection assay and Sanger sequencing. We found that the repair of the on-and off-target cleavage resulted in a wide variety of insertions, deletions and point mutations. Therefore, CRISPR/Cas9 systems need to be carefully designed to avoid potential off-target cleavage sites, including those with mismatches to the 12-bases proximal to the guide strand protospacer-adjacent motif.