The DOI matches its Crossref record; 43 of the 44 references it cites were found in a registry or an index.
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
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“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]
Experimental — accuracy not yet measuredThe cited source says what the claim attributes to it.
In the citing paper
“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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Cites
The Sequence Alignment/Map format and SAMtools
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Fast and accurate short read alignment with Burrows-Wheeler transform
Li, H., & Durbin, R. (2009) · Bioinformatics · doi:10.1093/bioinformatics/btp324 · Cited by 58,607
Multiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339
Fast and accurate long-read alignment with Burrows-Wheeler transform.
Li, H., & Durbin, R. (2010) · Bioinformatics · doi:10.1093/bioinformatics/btp698 · Cited by 12,063
Genome-scale CRISPR-Cas9 knockout screening in human cells
Shalem, O., Sanjana, N. E., Hartenian, E., Shi, X., Scott, D. A., Mikkelsen, T. S., Heckl, D., Ebert, B. L., Root, D. E., Doench, J. G., et al. (2014) · Science · doi:10.1126/science.1247005 · Cited by 5,234
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity
Ran, F. A., Hsu, P. D., Lin, C.-Y., Gootenberg, J. S., Konermann, S., Trevino, A. E., Scott, D. A., Inoue, A., Matoba, S., Zhang, Y., et al. (2013) · Cell · doi:10.1016/j.cell.2013.08.021 · Cited by 3,299
CRISPR-Cas systems for editing, regulating and targeting genomes
Sander, J. D., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2842 · Cited by 3,153
High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method
Gietz, R. D., & Schiestl, R. H. (2007) · Nature Protocols · doi:10.1038/nprot.2007.13 · Cited by 2,785
In vivo genome editing using Staphylococcus aureus Cas9
Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., Zetsche, B., Shalem, O., Wu, X., Makarova, K. S., et al. (2015) · Nature · doi:10.1038/nature14299 · Cited by 2,687
High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects
Kleinstiver, B. P., Pattanayak, V., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Zheng, Z., & Joung, J. K. (2016) · Nature · doi:10.1038/nature16526 · Cited by 2,673
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1 of the 44 references couldn’t be matched to a known paper.
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Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors
Anzalone, A. V., Koblan, L. W., & Liu, D. R. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0561-9 · Cited by 2,237
The next generation of CRISPR–Cas technologies and applications
Pickar-Oliver, A., & Gersbach, C. A. (2019) · Nature Reviews Molecular Cell Biology · doi:10.1038/s41580-019-0131-5 · Cited by 1,459
CRISPR technology: A decade of genome editing is only the beginning
Wang, J. Y., & Doudna, J. A. (2023) · Science · doi:10.1126/science.add8643 · Cited by 1,105
Vakulskas, C. A., Dever, D. P., Rettig, G. R., Turk, R., Jacobi, A. M., Collingwood, M. A., Bode, N. M., McNeill, M. S., Yan, S., Camarena, J., et al. (2018) · Nature Medicine · doi:10.1038/s41591-018-0137-0 · Cited by 782
CRISPR/Cas9 therapeutics: progress and prospects
Li, T., Yang, Y., Qi, H., Cui, W., Zhang, L., Fu, X., He, X., Liu, M., Li, P., & Yu, T. (2023) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-023-01309-7 · Cited by 824
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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