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PreprintCited by 15CRISPR and Genetic EngineeringRNA regulation and diseaseEnzyme Catalysis and Immobilization

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Eukaryotic-driven directed evolution of Cas9 nucleases

Open access
Ruta, Giulia Vittoria, Ciciani, Matteo, Kheir, Eyemen, Gentile, Michele Domenico, Amistadi, Simone, Casini, Antonio, Cereseto, Anna·2023·Genome biology
DOI 10.1101/2023.09.18.558227PMC PMC10962177

Preprint — published as 10.1186/s13059-024-03215-9

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Abstract

Full exploitation of the natural reservoir of CRISPR-Cas nucleases from prokaryotes for genome editing is limited by the suboptimal activity of these enzymes in mammalian cells. Here we developed a E ukaryotic P latform to Improve C as A ctivity (EPICA) to steer weakly active Cas9 nucleases into highly active enzymes by directed evolution. The EPICA platform is obtained by coupling Cas nuclease activity with yeast auxotrophic selection followed by mammalian cell selection through a sensitive reporter system. EPICA was validated with a poorly efficient Cas9 nuclease from Campylobacter jejuni , CjCas9, generating an enhanced variant, UltraCjCas9, following directed evolution rounds. UltraCjCas9 was up to 12-fold more active in mammalian endogenous genomic loci, while preserving high genome-wide specificity. Here we report a eukaryotic pipeline allowing enhancement of Cas9 systems, setting the ground to unlock the multitude of RNA-guided nucleases existing in nature.

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Metadata

Authors
Ruta, G. V., Ciciani, M., Kheir, E., Gentile, M. D., Amistadi, S., Casini, A., & Cereseto, A.
Affiliations
University of Trento (IT)
Corresponding author
  • Ruta, Giulia Vittoria
  • Cereseto, Anna
ORCID
Venue
Genome biology
Publisher
openRxiv
Published
2023-09-18
Volume
25
Issue
1
Pages
79-79
ISSN
1465-6906 (ISSN-L)
DOI
10.1101/2023.09.18.558227
PMC
PMC10962177
OpenAlex
W4393159098
Cited by
15
References
1
Citations by year
2026: 3 · 2025: 8 · 2024: 4
FWCI
2.04
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Genome biology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Institutional Research Information System (Università degli Studi di Trento) (submittedVersion, cc-by)
  • HAL (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • European Commission (HORIZON2020, 825825)
  • Università degli Studi di Trento
  • HORIZON EUROPE Framework Programme (825825)
  • Horizon 2020 (825825)
  • HORIZON EUROPE European Innovation Council (01071041)
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 › Enzyme Catalysis and Immobilization
Keywords
Cas9 nuclease, directed evolution, genome editing, reporter system, RNA-guided nucleases
MeSH terms
Gene Editing, Animals, Mammals, Genome, CRISPR-Cas Systems

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