Fully Modified SpyCas9 Guide RNAs Enable Robust Genome Editing In Cells and In Vivo

Area of Science:

• Molecular Biology
• Biotechnology
• Genetic Engineering

Background:

• CRISPR/Cas9 technology is crucial for genome editing.
• Chemically stabilized guide RNAs (gRNAs) offer potential for improved in vivo editing and delivery.
• Previous fully modified gRNAs showed reduced Cas9 activity.

Purpose of the Study:

• To optimize chemically stabilized guide RNAs (gRNAs) for CRISPR/Cas9.
• To enhance in vivo genome editing efficacy and delivery flexibility.
• To overcome reduced Cas9 activity associated with modified gRNAs.

Main Methods:

• Iterative, structure-guided optimization of SpyCas9 gRNAs.
• Systematic introduction of chemical modifications at each nucleotide position.
• Incorporation of 2'-amino-RNA, 4'-thio-RNA, and extended nucleic acid (exNA).

Main Results:

• Developed gRNA designs with 90-100% sugar- or backbone-modified nucleotides.
• Established modified gRNAs that maintain or enhance editing efficacy in vitro and in vivo.
• Observed sequence-dependent variability in modification patterns.

Conclusions:

• Heavily and fully modified gRNAs show promise for CRISPR/Cas9 applications.
• Optimized gRNAs can improve genome editing tools like nuclease and base editing.
• These modified gRNAs offer potential for advanced therapeutic genome editing.