Biased and unbiased methods for the detection of off-target cleavage by CRISPR/Cas9: An overview
Open accessAbstract
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 endonuclease (Cas9) derived from bacterial adaptive immune systems is a revolutionary tool used in both basic and applied science. It is a versatile system that enables the genome of different species to be modified by generating double strand breaks (DSBs) at specific locations. However, all of the CRISPR/Cas9 systems can also produce DSBs at off-target sites that differ substantially from on-target sites. The generation of DSBs in locations outside the intended site can produce mutations that need to be carefully monitored, especially when using these tools for therapeutic purposes. However, off-target analyses of the CRISPR/Cas9 system have been very challenging, particularly when performed directly in cells. In this manuscript, we review the different strategies developed to identify off-targets generated by CRISPR/cas9 systems and other specific nucleases (ZFNs, TALENs) in real target cells.
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IDLV has low sensitivity and a high false positive rate, probably due to non-specific IDLV integration and PCR amplification.
“The disadvantages of IDLV include its low sensitivity and high false positive rate, probably due to non-specific IDLV integration and PCR amplification (Martin et al., 2016).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Martin et al., 2016
Section 3.2 (page unavailable) says IDLV capture detects off-target cleavage at a frequency of 1% and that many IDLV insertions occur at non-relevant genomic sites, requiring controls. Section 3.3 (page unavailable) describes GUIDE-seq detecting a lower frequency of 0.1%, supporting a relative sensitivity limitation. The paper does not attribute a high false-positive rate to PCR amplification or quantify such a rate for IDLV. (LLM, conf=0.84)
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Cites
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“Although whole-genome sequencing can remediate the limitation, it is associated with high costs [83].”
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“However, none of the methods for detecting the distribution and frequency of off-targets introduced by SENs are regarded as sufficiently robust to be implemented in clinical trials.(14) Nevertheless, it is very useful to compare different off-target SEN activities in order to develop more effective and safer strategie…”
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“Differences among the several off-target detection methods are outside the scope of this manuscript, but several reviews extensively describe the available tools [140,141].”
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CRISPR GENOME SURGERY IN THE RETINA IN LIGHT OF OFF-TARGETING
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“GUIDE-seq is considered one of the most sensitive methods for detecting off-target effects in human cells [50].”
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“9,17 The same methodology allowed rAAV6 donor-mediated homologous recombination in the b -globin encoding and MyD88 loci 18 and also in the pyruvate kinase L/R locus for the treatment of pyruvate kinase de fi ciency.”
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“The capacity of IDLVs to integrate into DNA DSBs has been used by several research teams for unbiased detection of off-target sites generated by endonucleases [22], such as zinc finger nucleases (ZFNs) [23], the CRISPR/Cas9 system and transcription activator-like effector nucleases (TALENs) [24].”
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CRISPR off-target analysis platforms
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“20 As an unbiased approach, WGS provides a comprehensive database to characterize the off-target effects better than Sanger or targeted deep sequencing.”
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CRISPR/CAS as a Powerful Tool for Human Immunodeficiency Virus Cure: A Review
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“66 67–69 This may be attributed to the compromised functionality of DNA repair pathways in tumor cells, leading to an elevated incidence of off-target effects.”
Applications of CPPs in Genome Editing of Plants
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“We have considered only the sequences s i , instead of the entire genome, as the regions for off-target analysis known as biased off-target analysis [42].”
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Advances in Off-Target Detection for CRISPR-Based Genome Editing
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“25 The 4 nucleotides (nt) at positions + 4 to + 7 upstream of the 3 ¢ end PAM are highly sensitive to target mismatches.”
Evidence synthesis on the impact of genome editing on plant breeding
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The Need for Transfer Learning in CRISPR-Cas Off-Target ScoringPreprint
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“This method is highly relevant for genotoxicity assessment, as it offers a more accurate estimate of the genomic instability caused by editing events [34,35].”
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CRISPR Gene Editing of Hematopoietic Stem and Progenitor Cells
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CRISPR: A Promising Toolbox for Better Crops
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“Methods for the identification of CRISPR-Cas9 off-target sites have been developed and are reviewed elsewhere [31].”
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“It is known that other off-target modifications may occur in places not predicted in the candidate list (Table 6) due to a variety of reasons including genomic and epigenomic properties that may affect cleavage frequency; therefore, unbiased detection of off-target cleavage such as whole genome sequencing is recommend…”
BackgroundCRISPR driven Cyanobacterial Metabolic Engineering and its role in metabolite production
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“CRISPR‐Cas9 may also contribute to the future development of personalized cancer therapies by enabling therapeutic strategies tailored to tumor‐specific genetic alterations [155–157].”
- Authors
- Martin, F., Sánchez-Hernández, S., Gutiérrez-Guerrero, A., Pinedo-Gomez, J., & Benabdellah, K.
- Affiliations
- Genomic Medicine Department, GENYO—Centre for Genomics and Oncological Research Pfizer-Universidad de Granada-Junta de Andalucía, Avda de la Ilustración 114, 18007 Granada, Spain
- Universidad de Granada (ES)
- Corresponding author
- Benabdellah, Karim
- Venue
- International Journal of Molecular Sciences
- Publisher
- MDPI AG
- Published
- 2016-09-08
- Volume
- 17
- Issue
- 9
- Pages
- 1507
- ISSN
- 1422-0067 (ISSN-L)
- PMC
- PMC5037784
- OpenAlex
- W2515083702
- Cited by
- 92
- References
- 39
- Citations by year
- 2026: 5 · 2025: 3 · 2024: 10 · 2023: 9 · 2022: 9 · 2021: 16 · 2020: 16 · 2019: 8 · 2018: 12 · 2017: 3 · 2016: 1
- FWCI
- 0.72
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- International Journal of Molecular Sciences (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- RISalud Institutional Health Repository of Andalucía (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Fondo de Investigaciones Sanitarias ISCIII (PI12/01097, PI15/02015)
- Consejería de Economía, Innovación, Ciencia y Empleo (P09-CTS-04532, PI-57069, PI-0001/2009)
- 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
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Keywords
- CRISPR/Cas9, off-target cleavage, off-target detection, DNA double-strand breaks, genome editing, zinc finger nucleases, TALEN
- MeSH terms
- Gene Editing, Animals, Humans, Bias, Gene Targeting, CRISPR-Cas Systems
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