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High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
Open accessAbstract
The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.
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“Moreover, in-depth studies on the outcomes of Cas9-induced double-strand break (DSB) repair have shown significant off-target effects, which resulted in substitution, insertion, deletion, and DNA translocation.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [12]
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Genome engineering using the CRISPR-Cas9 system
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“We and others have previously shown that, although singlebase mismatches between the PAM-distal region of the guide sequence and target DNA are well tolerated by Cas9, multiple mismatches in this region can significantly affect on-target activity (Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013a; Pattanayak et al…”
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“The dosage of CRISPR/Cas9 is another factor affecting off-target mutations and should be carefully controlled (29,78).”
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“For example, the cleavage activity induced by the R-01 variant at position 2/1 may be alternatively interpreted as Cas9 cleavage with a GTG PAM (Figure 2C and Supplementary Figure S1), which is highly unlikely according to previous studies (20,21).”
Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System
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“Previous studies in human cell lines indicated a high frequency of off-target effect of CRISPR/Cas-induced mutagenesis (12, 13) but a lower off-target effect in mice and zebrafish (14, 15).”
BackgroundGene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA
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“Recently, a number of reports have systematically examined specificity of the CRISPR/Cas system in human cells as well as in vitro [26-30].”
BackgroundHighly Improved Gene Targeting by Germline-Specific Cas9 Expression in Drosophila
Kondo, S., & Ueda, R. (2013) · Genetics · doi:10.1534/genetics.113.156737 · Cited by 580
“Recent studies have shown that Cas9 accommodates multiple mismatches in the 20-bp target sequence, raising concerns about off-target effect (Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013).”
BackgroundMultiplexed CRISPR technologies for gene editing and transcriptional regulation
McCarty, N. S., Graham, A. E., Studená, L., & Ledesma‐Amaro, R. (2020) · Nature Communications · doi:10.1038/s41467-020-15053-x · Cited by 567
Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents
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Genome Engineering with Targetable Nucleases
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Editing plant genomes with CRISPR/Cas9
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Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9
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“It has also been shown that the strength of the promoter can have an effect on target specificity, and a high concentration of sgRNACas9 transcripts can increase off-target mutations (Hsu et al. 2013; Pattanayak et al. 2013).”
BackgroundRNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection
Hu, W., Kaminski, R., Yang, F., Zhang, Y., Cosentino, L., Li, F., Luo, B., Alvarez-Carbonell, D., Garcia-Mesa, Y., Karn, J., et al. (2014) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1405186111 · Cited by 527
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“Three independent studies have shownCRISPR/Cas9 could inducemutations even at the sites with up to five mismatches (Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013).”
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“Recent studies suggest that the CRISPR/Cas system may have offtarget effects (Cho et al., 2014; Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013a; Pattanayak et al., 2013), potentially confounding genetic studies in hPSCs.”
BackgroundIn vivo base editing rescues Hutchinson–Gilford progeria syndrome in mice
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Mapping the genomic landscape of CRISPR-Cas9 cleavage
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“Of note, the on-target site was recovered with the lowest concentration of nuclease that permitted recovery of target sites, most often at 0.25 nM sgRNP ( Supplementary Tables 2 – 9 ).”
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“…been demonstrated to produce off-target effects, i.e., mutagenesis at sites in the genome other than the desired on-target site (Hockemeyer et al., 2011; Mussolino et al., 2011; Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013a; Pattanayak et al., 2013; Cradick et al., 2013; Cho et al., 2014).”
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“This corroborates previous studies showing that lower levels of Cas9 and sgRNA can reduce off-target effects 14, 16 .”
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Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing
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Programmable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems
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DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing
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Small molecule–triggered Cas9 protein with improved genome-editing specificity
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Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae
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Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities
van Dongen, J. E., Berendsen, J. T. W., Steenbergen, R. D. M., Wolthuis, R. M. F., Eijkel, J. C. T., & Segerink, L. I. (2020) · Biosensors and Bioelectronics · doi:10.1016/j.bios.2020.112445 · Cited by 424
“It was shown for Cas9 that less than 5% of crRNA sequences with two or more mismatches to the target sequence is functional in target binding (Anderson et al., 2015) and mismatches near the PAM region are less tolerated (Pattanayak et al., 2013).”
COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites
Cradick, T. J., Qiu, P., Lee, C. M., Fine, E. J., & Bao, G. (2014) · Molecular Therapy - Nucleic Acids · doi:10.1038/mtna.2014.64 · Cited by 408
Expanding the Biologist’s Toolkit with CRISPR-Cas9
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“, 2013), leading many laboratories to analyze cleavage specificity more thoroughly using different approaches (Hsu et al., 2013; Mali et al., 2013a; Pattanayak et al., 2013).”
BackgroundUnraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach
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Naturally Occurring Off-Switches for CRISPR-Cas9
Pawluk, A., Amrani, N., Zhang, Y., García, B., Hidalgo-Reyes, Y., Lee, J., Edraki, A., Shah, M. K., Sontheimer, E. J., Maxwell, K. L., et al. (2016) · Cell · doi:10.1016/j.cell.2016.11.017 · Cited by 392
“For example, offtarget effects (cleavage and mutation at unintended, nearcognate genomic sites) are exacerbated by excessive or prolonged Cas9 activity (Fu et al., 2014; Hsu et al., 2013; Pattanayak et al., 2013).”
Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system
Sakuma, T., Nishikawa, A., Kume, S., Chayama, K., & Yamamoto, T. (2014) · Scientific Reports · doi:10.1038/srep05400 · Cited by 352
Metadata
- Authors
- Pattanayak, V., Lin, S., Guilinger, J. P., Ma, E., Doudna, J. A., & Liu, D. R.
- Affiliations
- Howard Hughes Medical Institute (US)
- Corresponding author
- Doudna, Jennifer A
- Liu, David R
- Venue
- Nature Biotechnology
- Published
- 2013-08-11
- Volume
- 31
- Issue
- 9
- Pages
- 839-843
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC3782611
- OpenAlex
- W2149402050
- Cited by
- 1,487
- References
- 21
- Citations by year
- 2026: 29 · 2025: 62 · 2024: 81 · 2023: 72 · 2022: 113 · 2021: 136 · 2020: 144 · 2019: 109 · 2018: 127 · 2017: 145 · 2016: 149 · 2015: 135 · 2014: 152 · 2013: 28
- FWCI
- 69.33
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- Howard Hughes Medical Institute
- National Institute of General Medical Sciences (R01GM073794, T32GM007753)
- 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, seed sequence, in vitro selection, genome editing, PAM sequence, HEK293T cells, human genome
- MeSH terms
- Bacterial Proteins, DNA, Endonucleases, Genetic Engineering, Humans, Streptococcus pyogenes, Genome, Sequence Analysis, DNA, Genomics, HEK293 Cells, RNA, Small Untranslated, High-Throughput Nucleotide Sequencing
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