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A highly specific SpCas9 variant is identified by in vivo screening in yeast
Open accessAbstract
Despite the utility of CRISPR-Cas9 nucleases for genome editing, the potential for off-target activity limits their application, especially for therapeutic purposes. We developed a yeast-based assay to identify optimized Streptococcus pyogenes Cas9 (SpCas9) variants that enables simultaneous evaluation of on- and off-target activity. We screened a library of SpCas9 variants carrying random mutations in the REC3 domain and identified mutations that increased editing accuracy while maintaining editing efficiency. We combined four beneficial mutations to generate evoCas9, a variant that has fidelity exceeding both wild-type (79-fold improvement) and rationally designed Cas9 variants (fourfold average improvement), while maintaining near wild-type on-target editing efficiency (90% median residual activity). Evaluating evoCas9 on endogenous genomic loci, we demonstrated a substantially improved specificity and observed no off-target sites for four of the eight single guide RNAs (sgRNAs) tested. Finally, we showed that following long-term expression (40 d), evoCas9 strongly limited the nonspecific cleavage of a difficult-to-discriminate off-target site and fully abrogated the cleavage of two additional off-target sites.
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“Meanwhile, the absolute on-target activity of these Cas9 mutants are not dramatically reduced as compared to SpCas9 (Casini et al., 2018).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Casini et al., 2018
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“CRISPR-nucleases have been successfully used to induce NHEJ to knockout genes or genomic regulatory elements25,26. This includes permanent repair of splicing defects in CFTR minigene models27 as an alternative to the transient and inefficient use of oligonucleotides or spliceosome-mediated RNA trans-splicing (SMaRT) strategies28,29.”
From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [26]
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“This screen identified four key beneficial point mutations in SpCas9, giving rise to evoCas9, a variant that exhibits fidelity exceeding SpCas9-HF1 and eSpCas9 (Kleinstiver et al., 2015; Casini et al., 2018).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Casini et al., 2018
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“They found that the number of off-target sites reduced by 98.7%, 95.4% and 94.1%, respectively.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Casini et al., 2018
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“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 [20]
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“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 [20]
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Metadata
- Authors
- Casini, A., Olivieri, M., Petris, G., Montagna, C., Reginato, G., Maule, G., Lorenzin, F., Prandi, D., Romanel, A., Demichelis, F., Inga, A., & Cereseto, A.
- Affiliations
- University of Trento (IT)
- Corresponding author
- Casini, Antonio
- Cereseto, Anna
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2018-01-29
- Volume
- 36
- Issue
- 3
- Pages
- 265-271
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC6066108
- OpenAlex
- W2787206763
- Cited by
- 493
- References
- 44
- Citations by year
- 2026: 33 · 2025: 48 · 2024: 65 · 2023: 43 · 2022: 82 · 2021: 62 · 2020: 70 · 2019: 63 · 2018: 26
- FWCI
- 19.66
- 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)
- Institutional Research Information System (Università degli Studi di Trento) (submittedVersion, other-oa)
- Institutional Research Information System (University of Udine) (submittedVersion)
- Funders
- Università degli Studi di Trento
- 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
- SpCas9, off-target effects, editing specificity, yeast-based screening, single guide RNA, genome editing, CRISPR/Cas9
- MeSH terms
- Gene Editing, CRISPR-Associated Protein 9, RNA, Guide, CRISPR-Cas Systems, Mutation, Streptococcus pyogenes, Substrate Specificity, RNA, Guide, Kinetoplastida, CRISPR-Cas Systems
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