Eukaryotic-driven directed evolution of Cas9 nucleases.
Journal
Genome biology
ISSN: 1474-760X
Titre abrégé: Genome Biol
Pays: England
ID NLM: 100960660
Informations de publication
Date de publication:
25 Mar 2024
25 Mar 2024
Historique:
received:
26
09
2023
accepted:
13
03
2024
medline:
26
3
2024
pubmed:
26
3
2024
entrez:
26
3
2024
Statut:
epublish
Résumé
Further advancement of genome editing highly depends on the development of tools with higher compatibility with eukaryotes. A multitude of described Cas9s have great potential but require optimization for genome editing purposes. Among these, the Cas9 from Campylobacter jejuni, CjCas9, has a favorable small size, facilitating delivery in mammalian cells. Nonetheless, its full exploitation is limited by its poor editing activity. Here, we develop a Eukaryotic Platform to Improve Cas Activity (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 is validated with CjCas9, generating an enhanced variant, UltraCjCas9, following directed evolution rounds. UltraCjCas9 is up to 12-fold more active in mammalian endogenous genomic loci, while preserving high genome-wide specificity. We report a eukaryotic pipeline allowing enhancement of Cas9 systems, setting the ground to unlock the multitude of RNA-guided nucleases existing in nature.
Sections du résumé
BACKGROUND
BACKGROUND
Further advancement of genome editing highly depends on the development of tools with higher compatibility with eukaryotes. A multitude of described Cas9s have great potential but require optimization for genome editing purposes. Among these, the Cas9 from Campylobacter jejuni, CjCas9, has a favorable small size, facilitating delivery in mammalian cells. Nonetheless, its full exploitation is limited by its poor editing activity.
RESULTS
RESULTS
Here, we develop a Eukaryotic Platform to Improve Cas Activity (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 is validated with CjCas9, generating an enhanced variant, UltraCjCas9, following directed evolution rounds. UltraCjCas9 is up to 12-fold more active in mammalian endogenous genomic loci, while preserving high genome-wide specificity.
CONCLUSIONS
CONCLUSIONS
We report a eukaryotic pipeline allowing enhancement of Cas9 systems, setting the ground to unlock the multitude of RNA-guided nucleases existing in nature.
Identifiants
pubmed: 38528620
doi: 10.1186/s13059-024-03215-9
pii: 10.1186/s13059-024-03215-9
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
79Subventions
Organisme : Horizon 2020
ID : 825825
Organisme : HORIZON EUROPE European Innovation Council
ID : 01071041
Informations de copyright
© 2024. The Author(s).
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