Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5′ G-extended sgRNAs
Open accessAbstract
Increased fidelity mutants of the SpCas9 nuclease constitute the most promising approach to mitigating its off-target effects. However, these variants are effective only in a restricted target space, and many of them are reported to work less efficiently when applied in clinically relevant, pre-assembled, ribonucleoprotein forms. The low tolerance to 5'-extended, 21G-sgRNAs contributes, to a great extent, to their decreased performance. Here, we report the generation of Blackjack SpCas9 variant that shows increased fidelity yet remain effective with 21G-sgRNAs. Introducing Blackjack mutations into previously reported increased fidelity variants make them effective with 21G-sgRNAs and increases their fidelity. Two "Blackjack" nucleases, eSpCas9-plus and SpCas9-HF1-plus are superior variants of eSpCas9 and SpCas9-HF1, respectively, possessing matching on-target activity and fidelity but retaining activity with 21G-sgRNAs. They facilitate the use of existing pooled sgRNA libraries with higher specificity and show similar activities whether delivered as plasmids or as pre-assembled ribonucleoproteins.
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Modifications at the 5′ end of the gRNA, such as addition of secondary structures or unpaired nucleotides, lower off-target activity.
“Modifications at the 5′end of the gRNA, such as the addition of secondary structures (Kocak et al., 2019) or unpaired nucleotides (Kulcsár et al., 2020), have also been found to lower off-target activity.”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Kulcsár et al., 2020
Current regulations may differentiate between genome editing techniques (SDN-1, SDN-2, SDN-3) and the use of foreign DNA or sequence templates in the editing process.
“Current regulations may differentiate between genome editing techniques (SDN-1, SDN-2, SDN-3) and the use of foreign DNA or sequence templates in the editing process (Kulcsár et al., 2020).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Kulcsár et al., 2020
The Introduction discusses Cas9 targeting and guide-RNA methods, while the Methods describe plasmid-based editing experiments. Our copy does not discuss regulations, SDN-1/2/3 classifications, or regulatory distinctions involving foreign DNA or sequence templates (searched for those terms and concepts). (LLM, conf=0.76)
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- Authors
- Kulcsár, P. I., Tálas, A., Tóth, E., Nyeste, A., Ligeti, Z., Welker, Z., & Welker, E.
- Affiliations
- University of Szeged (HU)
- Semmelweis University (HU)
- Institute of Molecular Life Sciences (HU)
- Laboratory for the Bioengineering of Tissues (FR)
- HUN-REN Szegedi Biológiai Kutatóközpont (HU)
- Corresponding author
- Kulcsár, Péter István
- Welker, Ervin
- Venue
- Nature Communications
- Published
- 2020-03-06
- Volume
- 11
- Issue
- 1
- Pages
- 1223-1223
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC7060260
- OpenAlex
- W3009572786
- Cited by
- 45
- References
- 63
- Citations by year
- 2026: 7 · 2025: 6 · 2024: 9 · 2023: 7 · 2022: 10 · 2021: 4 · 2020: 2
- FWCI
- 1.87
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nature Communications (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Repository of the Academy's Library (Library of the Hungarian Academy of Sciences) (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- National Research, Development and Innovation Office
- 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
- SpCas9, on-target activity, off-target effects, ribonucleoprotein delivery, CRISPR/Cas9 genome editing
- MeSH terms
- Gene Editing, CRISPR-Associated Protein 9, RNA, Guide, CRISPR-Cas Systems, Humans, Mutation, Structure-Activity Relationship, Mutagenesis, RNA, Guide, Kinetoplastida, Protein Structure, Secondary, Crystallography, X-Ray, HEK293 Cells, CRISPR-Cas Systems
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