Cited by 1,273CRISPR and Genetic EngineeringRNA regulation and diseaseRNA Interference and Gene Delivery

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Off-target Effects in CRISPR/Cas9-mediated Genome Engineering

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Zhang, Xiao-Hui, Tee, Louis Y, Wang, Xiao-Gang, Huang, Qun-Shan, Yang, Shi-Hua·2015·Molecular Therapy - Nucleic Acids

DOI 10.1038/mtna.2015.37PMC PMC4877446

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Abstract

CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)-RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site-is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype-phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology.

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Figure 1 Base preferences for CRISPR/Cas9 system. Cas9 protein is shown in orange. PAM and seed sequences are shown in red. Base preferences are shown in blue. Scissors indicate cleavage sites. The single-letter DNA code is used (N=A, T, C, G; R=G or A).CC BY-NC-SA · PubMed Central

Figure 2 Methods for optimal CRISPR/Cas9 system for biomedical and clinical application. (a) Double nicking by RNA-Guided CRISPR Cas9 for enhanced genome-editing speciﬁcity. Cas9 protein is shown in orange. Scissors indicate cleavage sites. (b) Fusion of catalytically inactive Cas9 to Fok I nuclease. (c) Delivery of Cas9 protein. cPP, cell-penetrating peptide. The single-letter codes for amino acids are used. C, cysteine; G, glycine; R, arginine; L, leucine. His, Histidine tag; HA, hemagglutinin tag; Mal, maleimide. (d) Alternative PAMs for CIRSPR/Cas9 system. (e) SgRNA design—CRISPR/Cas9 design tools. (f) Method of off-target detection-Degenome-seq. IGV, Integrative Genomics Viewer; WGS, whole-genome sequencing; WT, wild type.CC BY-NC-SA · PubMed Central

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Authors: Zhang, X.-H., Tee, L. Y., Wang, X.-G., Huang, Q.-S., & Yang, S.-H.
Affiliations: McGovern Institute for Brain Research (US)
ORCID: Tee, Louis Y
Venue: Molecular Therapy - Nucleic Acids
Publisher: Elsevier BV
Published: 2015
Volume: 4
Pages: e264
ISSN: 2162-2531 (ISSN-L)
DOI: 10.1038/mtna.2015.37
PMC: PMC4877446
OpenAlex: W2173737475
Cited by: 1,273
References: 82
Citations by year: 2026: 67 · 2025: 145 · 2024: 166 · 2023: 151 · 2022: 164 · 2021: 198 · 2020: 132 · 2019: 109 · 2018: 80 · 2017: 35 · 2016: 25 · 2015: 1
FWCI: 4.55
Type: journal article
Language: en
Open access: gold
doi.org
Licence: cc-by-nc-sa
www.elsevier.com/tdm/userlicense/1.0/ (tdm)
creativecommons.org/licenses/by-nc-sa/4.0/ (vor, 645 d)
Locations: Molecular Therapy — Nucleic Acids (publishedVersion, cc-by-nc-sa)
Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
PubMed Central (publishedVersion, cc-by-nc-sa)
PubMed (publishedVersion)
DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords: CRISPR/Cas9, off-target effects, genome editing, RNA-guided endonuclease, genome editing specificity, therapeutic genome editing

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Off-target Effects in CRISPR/Cas9-mediated Genome Engineering

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