CRISPR off-target analysis platforms
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)-Caspase9 (Cas9) system provides a programmable technology that may be used to edit the eukaryotic genome and epigenome. CRISPR/Cas9 includes a guide RNA targeted to a gene of interest which hybridizes to a nucleotide sequence next to a protospacer-adjacent motif (PAM) which guides the Cas9 endonucleases to the target site for cleavage via double-strand breaks. A caveat of the CRISPR/Cas9 system is the creation of off-target double-strand breaks (DSBs) which may result in anomalous insertions, deletions, and translocations. Thus, assays for the sensitive detection and analysis of off-target editing are critical. Here, we describe currently available CRISPR technologies, CRISPR applications, and current analysis platforms to detect off-target effects including genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-Seq), high-throughput genomic translocation sequencing (HTGTS), breaks labeling, enrichments on streptavidin and next-generation sequencing (BLESS), and in vitro nuclease-digested genome sequencing (Digenome-seq).
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LNP delivery in vivo can be quickly degraded.
“| Delivery methods improvement | Application in vivo [95–105] | LNP: Can be quickly degraded in vivo | Eliminate off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [96]
LNP delivery in vivo eliminates off-target effects.
“| Delivery methods improvement | Application in vivo [95–105] | LNP: Can be quickly degraded in vivo | Eliminate off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [96]
AAV delivery in vivo lasts for years in terminally differentiated cell types.
“| Delivery methods improvement | Application in vivo [95–105] | AAV: Last for years in terminally differentiated cell types | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [96]
AAV delivery in vivo may cause accumulation of off-target mutations.
“| Delivery methods improvement | Application in vivo [95–105] | AAV: Last for years in terminally differentiated cell types | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [96]
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Off-target effects in CRISPR/Cas9 gene editing
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“The advantage of this method is the ability to detect chromosomal translocations that may be missed by other methods (Hu et al., 2016; Li et al., 2019; Xu et al., 2023).”
- Authors
- Xu, C. L., Ruan, M. Z., Ragi, S. D., & Tsang, S. H.
- Affiliations
- Stanford Medicine (US)
- NewYork–Presbyterian Hospital (US)
- Brown University (US)
- Columbia University Irving Medical Center (US)
- Corresponding author
- Tsang, Stephen H.
- Venue
- Methods in molecular biology
- Publisher
- Springer US
- Published
- 2022-12-09
- Volume
- 2560
- Pages
- 279-285
- ISSN
- 1064-3745 (ISSN-L)
- 1940-6029
- OpenAlex
- W4311579433
- Cited by
- 6
- References
- 21
- Citations by year
- 2026: 1 · 2025: 3 · 2024: 1 · 2023: 1
- FWCI
- 2.10
- Type
- book chapter
- Language
- en
- Open access
- closed
- Licence
- Locations
- Methods in molecular biology (publishedVersion)
- PubMed (publishedVersion)
- 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 › DNA Repair Mechanisms
- Keywords
- CRISPR/Cas9, off-target effects, DNA double-strand breaks, off-target detection, GUIDE-seq, genome editing, protospacer adjacent motif, translocation, epigenome editing
- MeSH terms
- Genomics
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