Directed evolution of CRISPR-Cas9 to increase its specificity
Open accessPublished version of a preprint
Abstract
The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we develop a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without killing on-target activities in human cells. Unlike other engineered Cas9 variants, Sniper-Cas9 shows WT-level on-target activities with extended or truncated sgRNAs with further reduced off-target activities and works well in a preassembled ribonucleoprotein (RNP) format to allow DNA-free genome editing.
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In several previous reports, LZ3 Cas9, Sniper Cas9, and HiFi Cas9 had comparable editing activity to SpCas9-WT in human cells or other animal embryos.
“It worth mentioning that in several previous reports, LZ3 Cas9, Sniper Cas9, and HiFi Cas9 had comparable editing activity to the SpCas9-WT in human cells or other animal embryos.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [21]
The Results section (“Clone selection and characterization”; page unavailable) reports that Sniper-Cas9 had on-target activity comparable to WT-Cas9 in human HEK293T cells. Our copy does not establish the corresponding claim for LZ3 Cas9 or HiFi Cas9, or for animal embryos. (LLM, conf=0.88)
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- Authors
- Lee, J. K., Jeong, E., Lee, J., Jung, M., Shin, E., Kim, Y.-H., Lee, K., Jung, I., Kim, D., Kim, S., & Kim, J.-S.
- Affiliations
- ToolGen (South Korea) (KR)
- Seoul National University (KR)
- Corresponding author
- Lee, Jungjoon K.
- Lee, Joonsun
- Venue
- Nature Communications
- Published
- 2018-08-06
- Volume
- 9
- Issue
- 1
- Pages
- 3048-3048
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC6078992
- OpenAlex
- W2781931843
- Cited by
- 525
- References
- 40
- Citations by year
- 2026: 30 · 2025: 61 · 2024: 69 · 2023: 61 · 2022: 99 · 2021: 76 · 2020: 69 · 2019: 53 · 2018: 5
- FWCI
- 18.05
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nature Communications (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- Europe PMC (PubMed Central) (publishedVersion, other-oa)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Seoul National University Open Repository (Seoul National University) (submittedVersion)
- Funders
- National Research Foundation (2018M3A9H3020844, 2017M3A9B4061404)
- National Research Foundation of Korea (2017M3A9B4061404, 2017M3A9B4061406, 2018M3A9H3020844)
- Institute for Basic Science (IBSR021-D1, R021-D1)
- Ministry of Science and ICT, South Korea (2018M3A9H3020844)
- 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, directed evolution, off-target effects, DNA-free genome editing, single guide RNA, on-target activity, human cells
- MeSH terms
- Gene Editing, RNA, Guide, CRISPR-Cas Systems, DNA, Escherichia coli, Humans, Plasmids, Recombinant Proteins, Ribonucleoproteins, Substrate Specificity, RNA, Guide, Kinetoplastida, Directed Molecular Evolution, HEK293 Cells, CRISPR-Cas Systems
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