Rationally engineered Cas9 nucleases with improved specificity
Open accessAbstract
The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that "enhanced specificity" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.
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Chemical modification of gRNA by replacing RNA nucleotides with DNA nucleotides increases on-target efficiency and decreases off-target effects.
“| gRNA improvement | Chemical modifications [76–78] | Replacement of RNA nucleotides into DNA nucleotides | Increased on-target efficiency and decreased off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [77]
The paper reports reduced off-target effects, but attributes them to structure-guided engineering of the Cas9 protein, not replacement of guide-RNA nucleotides with DNA nucleotides. Its conclusion says on-target activity was preserved, not increased. The relevant passages are labeled Abstract in our copy; no page numbers are provided. (LLM, conf=0.86)
Experimental — accuracy not yet measuredhypaCas9 demonstrates higher on-target activity and lower off-target effects than eSpCas9 and SpCas9-HF1.
“Accordingly, scientists further designed hypaCas9 (hyper-accurate Cas9) (Chen et al., 2017), which demonstrates higher on-target activity and lower off-target effects than eSpCas9 and SpCas9-HF1 (Kleinstiver et al., 2016; Slaymaker et al., 2016; Chen et al., 2017).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Slaymaker et al., 2016
The indexed passage labeled Abstract (page not provided) identifies the eSpCas9 variants and reports that they reduced off-target effects while maintaining on-target activity. It does not assess hypaCas9 or SpCas9-HF1, so it cannot support the claimed superiority of hypaCas9 over either comparator. (LLM, conf=0.86)
Experimental — accuracy not yet measuredA series of optimized mutations in SpCas9 have greatly improved the editing specificity of Cas9 variants without substantially attenuating the editing activity.
“In addition, a series of optimized mutations in SpCas9 have greatly improved the editing specificity of Cas9 variants without substantially attenuating the editing activity.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [18]
Incorporation of MP/bridged nucleic acids/locked nucleic acids increases on-target efficiency and decreases off-target effects.
“Incorporation of MP/bridged nucleic acids/locked nucleic acids | Increased on-target efficiency and decreased off-target effects”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [77]
The paper’s Abstract reports that engineered Cas9 proteins reduce off-target effects while maintaining on-target cleavage. Our copy does not address incorporation of MP, bridged nucleic acids, or locked nucleic acids, or show that these modifications increase on-target efficiency. Searches for those terms and for chemically modified guide RNA found no supporting passage. (LLM, conf=0.76)
Experimental — accuracy not yet measuredSome active gRNAs for SpCas9-WT showed very low editing ability in SpCas9 variants.
“Some active gRNAs for SpCas9-WT showed very low editing ability in SpCas9 variants.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [18]
The paper’s on-target activity passage (indexed copy, section labeled Abstract; page unavailable) says the three variants produced indel levels similar to WT SpCas9 at the majority of 24 target sites. It does not say whether any guides active with WT had very low editing activity with the variants; the reported reductions elsewhere concern off-target cleavage or mismatched guides. Searched for guide-specific losses of on-target editing activity. (LLM, conf=0.68)
Experimental — accuracy not yet measured
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- Authors
- Slaymaker, I. M., Gao, L., Zetsche, B., Scott, D. A., Yan, W. X., & Zhang, F.
- Affiliations
- Broad Institute (US)
- Corresponding author
- Slaymaker, Ian M.
- Gao, Linyi
- Zhang, Feng
- Venue
- Science
- Published
- 2016-01
- Volume
- 351
- Issue
- 6268
- Pages
- 84-88
- ISSN
- 0036-8075 (ISSN-L)
- 1095-9203
- PMC
- PMC4714946
- OpenAlex
- W2188223075
- Cited by
- 2,414
- References
- 21
- Citations by year
- 2026: 116 · 2025: 171 · 2024: 168 · 2023: 172 · 2022: 250 · 2021: 226 · 2020: 283 · 2019: 266 · 2018: 249 · 2017: 266 · 2016: 236 · 2015: 5 · 2014: 2 · 2013: 2
- FWCI
- 109.62
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Science (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-sa)
- Funders
- National Institute of General Medical Sciences (T32GM007753)
- Daisy Soros Fellowship
- National Institutes of Health
- NIMH (5DP1-MH100706, 1R01MH110049)
- NIDDK (5R01DK097768-03)
- National Science Foundation
- Keck
- New York Stem Cell
- Damon Runyon
- Searle Scholars
- Merkin
- Vallee Foundations
- 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
- Cas9, CRISPR/Cas9, genome editing, off-target effects, off-target cleavage, protein engineering, structure-guided engineering, SpCas9, RNA-guided endonuclease, targeted deep sequencing, human cells
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Bacterial Proteins, Endonucleases, Humans, Protein Conformation, Streptococcus pyogenes, Protein Engineering, Mutagenesis, Point Mutation, RNA, Guide, Kinetoplastida, DNA Cleavage
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