Eukaryotic-driven directed evolution of Cas9 nucleases

Area of Science:

• Molecular Biology
• Biotechnology
• Gene Editing Technologies

Background:

• Advancements in genome editing necessitate tools with improved eukaryotic compatibility.
• Many Cas9 variants show potential but require optimization for efficient genome editing.
• The small size of Campylobacter jejuni Cas9 (CjCas9) is advantageous for delivery in mammalian cells, but its editing activity is limited.

Purpose of the Study:

• To develop a directed evolution platform for enhancing the activity of weakly active Cas9 nucleases.
• To create a more active and specific CjCas9 variant for eukaryotic genome editing applications.

Main Methods:

• Developed the Eukaryotic Platform to Improve Cas Activity (EPICA) using yeast auxotrophic selection and a mammalian cell reporter system.
• Employed directed evolution to enhance CjCas9 activity through iterative selection and screening.
• Validated the enhanced Cas9 variant in mammalian endogenous genomic loci.

Main Results:

• The EPICA platform successfully generated an enhanced CjCas9 variant, named UltraCjCas9.
• UltraCjCas9 exhibits up to 12-fold higher editing activity in mammalian cells compared to the wild-type CjCas9.
• The enhanced variant, UltraCjCas9, maintains high specificity across the genome.

Conclusions:

• A novel eukaryotic pipeline (EPICA) has been established for enhancing Cas9 nuclease activity.
• This platform facilitates the optimization of naturally occurring RNA-guided nucleases for genome editing.
• The development of UltraCjCas9 demonstrates the potential of EPICA to unlock new genome editing tools.