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

By Thomas J. Cradick, Eli J. Fine, C. Antico, Gang Bao. Nucleic Acids Research, 2013

629 citations recorded by the source corpus.

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.

Subfields: Molecular Biology, Plant Science

View Paper: https://www.semanticscholar.org/paper/a84b7e538ed06e01dc5bcaa01d9805cd0fd31461

How researchers see this paper

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In Molecular Biology

1. Jin-Soo Kim
2. Sojung Kim
3. S. Cho
4. Daesik Kim
5. Jungeun Kim
6. Ge Gao
7. Lei Kong
8. Jerilyn A. Timlin

In Plant Science

1. Ge Gao
2. S. Cho
3. Jin-Soo Kim
4. Jungeun Kim
5. Daesik Kim
6. Jerilyn A. Timlin
7. J. Carney
8. J. C. Harper

Citations in the corpus

• Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins (2014, background): “Recently, several groups independently reported that RGENs can cause unwanted mutations at off-target sites that differ by up to 5 nt from on-target sites, raising concerns about their specificities (Cradick et al. 2013; Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013).”
• CasOT: a genome-wide Cas9/gRNA off-target searching tool (2014, background): “For example, Cas9/gRNA seems to have considerable offtarget effects in cultured human cells (Cradick et al., 2013; Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013a; Pattanayak et al., 2013) and in several organisms (Fujii et al., 2013; Li et al., 2013; Shan et al., 2013).”
• Delivering CRISPR: a review of the challenges and approaches (2018, background): “This contrasts with many reports that show no detectable off-target cleavage from CRISPR/Cas9 editing, with off-target effects being guide-RNA-specific (Cradick et al., 2013; Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013;Cho et al., 2014).”
• Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila (2014, methodology): “Off-target cleavage has been raised as a serious concern for CRISPR/Cas9-mediated genome engineering (Cradick et al. 2013; Fu et al. 2013).”