GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases
Open accessAbstract
CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide, off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint 'hotspots'. Finally, GUIDE-seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced, off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases before clinical use.
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A primary limitation of GUIDE-seq is the low delivery efficiency of dsODNs into cells, resulting in detection of only 30%-50% of all DSBs.
“A primary limitation of GUIDE-seq is relevant to the low delivery efficiency of dsODNs into cells, which results in detection of only 30%–50% of all the DSBs (Tsai et al., 2015; Pan et al., 2022).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tsai et al., 2015
The Results (Overview of the GUIDE-Seq method; page unavailable) report robust dsODN integration, at rates two- to three-fold lower than indel frequencies at the tested sites—not detection of 30%–50% of all DSBs. The Discussion (page unavailable) describes dsODN integration as very efficient and reports detection of 38 of 40 previously identified off-target sites, contrary to the claimed primary limitation. (LLM, conf=0.87)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredGUIDE-seq off-target profiling of AsCas12a-crRNA+11 in HEK293/3272-26A>G cells showed exclusive editing of the 3272-26A>G CFTR locus, with no detectable non-specific cleavages in the second allele or any other genomic loci.
“Off-target profiling of AsCas12a-crRNA+11 genome editing in HEK293/3272-26A>G cells18,42 showed very high specificity, as demonstrated by exclusive editing of the 3272-26A>G CFTR locus, while non-specific cleavages in the second allele, or any other genomic loci, could not be detected (Fig. 2c and Supplementary Fig. 4c).”
From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [42]
The Discussion identifies GUIDE-seq as a method for assessing genome-wide off-target effects, supporting the method attribution. But the paper does not report profiling AsCas12a-crRNA+11 in HEK293/3272-26A>G cells or the claimed exclusive CFTR editing and absence of detectable cleavage elsewhere; its reported experiments concern other guide RNAs. (LLM, conf=0.84)
Experimental — accuracy not yet measuredIt defines off-target effects as cleavage at guide-mismatched sites and says they should be minimized for therapeutic editing.
“While cleavage at sites with perfect complementarity to the protospacer domain of the gRNA typically occurs with highest efficiency, cleavage at sites having one to several bases of mismatch with the gRNA can occur. These undesired cleavage events are called “off-target effects” (OTEs)10, and their existence should be minimized for therapeutic applications of genome editing technologies.”
From A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells (2018) · cites this paper as [10]
It describes GUIDE-seq as a method for empirically finding actual off-target sites in cells.
“The GUIDE-seq procedure10 can be used to empirically identify real off-target sites in cells.”
From A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells (2018) · cites this paper as [10]
GUIDE-seq can detect off-target sites with indel frequencies as low as 0.03%.
“GUIDE-seq can detect off-target sites with indel frequencies as low as 0.03% (Tsai et al., 2015).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tsai et al., 2015
GUIDE-seq is more sensitive than the IDLV method because dsODNs integrate more efficiently and precisely into DSBs, while IDLV integration events are low in number and can distribute as far as 500 bp away from the actual DSB sites.
“GUIDE-seq is more sensitive than the IDLV method because dsODNs integrate more efficiently and precisely into DSBs, while the integration events of IDLV is low in number and can distribute as far as 500bp away from the actual DSB sites (Tsai et al., 2015; Cromer et al., 2023).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tsai et al., 2015
Other publications observed a much lower validation rate for dCas9 ChIP-Seq.
“However, other publications observed a much lower validation rate (Cencic et al., 2014; Duan et al., 2014; Tsai et al., 2015).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tsai et al., 2015
Cas9 could associate with genomic loci without exerting its nuclease function, which is a major caveat in Cas9 ChIP-Seq-based off-target detection assays.
“These studies indicated that Cas9 could associate with genomic loci without exerting its nuclease function, deposing a major caveat in Cas9 ChIP-Seq-based off-target detection assay.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tsai et al., 2015
Extru-seq exhibited a much lower false negative rate than GUIDE-seq (2.3% versus 29%).
“Interestingly, compared to cell-based methods such as GUIDE-seq (see next section) (Tsai et al., 2015), Extru-seq exhibited much lower false negative rate (2.3% versus 29%) (Kwon et al., 2023).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tsai et al., 2015
In vitro Guide-Seq detected only the Fah target site and one off-target site (GOT1).
“Using the standard Guide-seq protocol, we detected only the Fah target site and one off-target site (defined as Guide-seq off-target site 1, or GOT1) (Supplementary Data Set 1).”
From Adenine base editing in an adult mouse model of tyrosinaemia (2019) · cites this paper as [23]
Silicon methods can only identify part of off-target cleavage.
“However, silicon methods can only identify part of the off-target cleavage.48”
From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [48]
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- Authors
- Tsai, S. Q., Zheng, Z., Nguyen, N. T., Liebers, M., Topkar, V. V., Thapar, V., Wyvekens, N., Khayter, C., Iafrate, A. J., Le, L. P., Aryee, M. J., & Joung, J. K.
- Affiliations
- Harvard University (US)
- Massachusetts General Hospital (US)
- Corresponding author
- Tsai, Shengdar Q
- Joung, J Keith
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2014-12-16
- Volume
- 33
- Issue
- 2
- Pages
- 187-197
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC4320685
- OpenAlex
- W2003797386
- Cited by
- 2,268
- References
- 39
- Citations by year
- 2026: 189 · 2025: 211 · 2024: 196 · 2023: 191 · 2022: 208 · 2021: 211 · 2020: 231 · 2019: 186 · 2018: 187 · 2017: 177 · 2016: 172 · 2015: 105 · 2014: 1
- FWCI
- 62.14
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- Funders
- National Institute of General Medical Sciences (DP1GM105378, R01GM088040, F32GM105189)
- National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR063070)
- National Center for Chronic Disease Prevention and Health Promotion (DP1 GM105378)
- 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
- GUIDE-seq, CRISPR-Cas nucleases, off-target cleavage, DNA double-strand breaks, genome-wide characterization, genome editing safety
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Cell Line, Humans, Oligodeoxyribonucleotides, Genome, Human, RNA Editing, DNA Breaks, Double-Stranded, High-Throughput Nucleotide Sequencing, CRISPR-Cas Systems
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