A eukaryotic platform for the enhancement of Cas nucleases

A large repertoire of CRISPR-Cas systems from bacteria, archaea and phages is available for a large variety of genome editing applications. Nevertheless, a limited number of orthologs have the same editing efficiency as SpCas9 in mammalian cells. Moreover, Cas with reduced molecular size that have been characterized and could give an advantage in delivery, are poorly active in human cells. Directed evolution was proved as a valid approach to identify Cas proteins with altered functions, however unbiased screenings to enhance Cas nucleases have been described only in bacterial cells. Here we develop a fully eukaryotic platform to evolve inefficient Cas proteins toward highly active nucleases. A first step of directed evolution is performed in yeast to isolate enhanced variants by auxotrophic selection; a further screening of the mutants directly in mammalian cells is achieved through a fluorescence activation system. We have first applied the platform to the smallest Type II Cas9 ortholog, CjCas9 (984aa): this double screening for improved activity led to the identification of the enhanced multiple mutant UltraCjCas9. Our variant showed increased efficiency in comparison with CjCas9 WT and the rational designed mutant enCjCas9, while maintaining similar precision editing profile. Several modifications of the screening platform were applied to evolve another small Cas, the hypercompact CasΦ2 from phages: yeast and mammalian cells steps were optimized resulting in a platform suitable for the enhancement of almost inactive Cas nucleases. Our directed evolution approach to enhance Cas nucleases activity in eukaryotic cells enables the improvement of these enzymes directly within the chromatin environment, expanding the toolbox of available genome editors. Moreover, mutations identified applying this screening can give an insight into protein residues or interactions with key roles for efficiency in human cells.

Grantor dc:publisher

Università degli studi di Trento

Year dc:date

2024

Author dc:creator

• Ruta, Giulia Vittoria

Contributors dc:contributor

• Cereseto, Anna

Statement dc:rights

• info:eu-repo/semantics/openAccess
• license:Tutti i diritti riservati (All rights reserved)
• license uri:iris.PRI01

Language dc:language

eng

Identifier

http://dx.doi.org/10.15168/11572_406870
10.15168/11572_406870

OAI identifier oai:identifier

oai:iris.unitn.it:11572/406870