bioRxiv : the preprint server for biologyJun 5, 2026

Fully Modified SpyCas9 Guide RNAs Allow Strong Genome Editing in Cells and Living Organisms

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Jun 27, 2026

Abstract

Essence

Structure-guided chemical optimization produced heavily and fully modified SpyCas9 guide RNAs that can maintain or improve genome editing activity.

Evidence

Platform optimization experiments systematically modified SpyCas9 gRNAs and tested 90-100% sugar- or backbone-modified guides in vitro and in vivo.

Caveat

Some modification patterns showed sequence-dependent variability, so performance is not uniform across all guide designs.

Simplified

Precision engineering of CRISPR/Cas9 components has advanced genome editing toward therapeutic applications. Completely chemically stabilized guide RNAs (gRNAs) have the potential to improveediting efficacy while enabling greater flexibility in delivery strategies. However, previous generations of fully modified guides have been associated with reduced Cas9 activity. Here, we employed an iterative, structure-guided optimization strategy to systematically introduce chemical modifications at each position of SpyCas9 gRNAs. Extending beyond commonly used nucleotide modifications, we incorporated 2'-amino-RNA, 4'-thio-RNA, and extended nucleic acid (exNA) to generate gRNA designs in which 90-100% of the nucleotides are sugar- or backbone-modified. Although certain modification patterns exhibit sequence-dependent variability, we have established a growing repertoire of guides that consistently maintain or enhance editing efficacy when applied bothand. Collectively, our heavily and fully modified gRNAs hold potential for applications in nuclease editing, base editing, and other genome editing tools. in vivo in vitro in vivo

Full Text

Full text is available at the source.

View Full Text ↗

Featured in

CRISPR Gene EditingIssue #40

90% of CRISPR guide RNA modified with new chemistry maintains full gene editing power

↗

Fully Modified SpyCas9 Guide RNAs Allow Strong Genome Editing in Cells and Living Organisms

Abstract

Full Text

Featured in

You found one interesting study. We’ll send the next 7.

what lands in your inbox each week:

Recent issues from the crispr gene editing brief