Abstract
Purpose :
CRISPR-Cas9 is a powerful tool for genomic manipulation. Its action is directed by a target-specific guide RNA (gRNA). Chemical modification of Streptococcus Pyogenes Cas9 (SpCas9) gRNA improves target binding affinity, and enhances knockdown specificity and efficacy. However, excessive modification abolishes SpCas9 activity. All Cas9 species and engineered mutants require a gRNA to direct action. We therefore aimed to make similar modifications to Staphylococcus Aureus (SaCas9) gRNA to investigate if the principles of chemical modification could be applied to other gRNAs.
Methods :
Using the crystal structure of SaCas9 bound to its gRNA as a guide, we created several gRNAs: 1) an “unmodified” gRNA 2) an “ends modified” gRNA where the 3 terminal nucleotides were modified with 2’O-methylation and phosphothioated bonds 3) a “loops modified” gRNA where gRNA “loops” were modified 4) a “heavily modified” sgRNA where all non-interacting gRNA nucleotides and the gRNA tail was fully modified 5) a “heavily modified body” gRNA modified as (4) but without tail modifications 6) a “heavily modified tail” sgRNA modified as (4) but without body modifications and finally 7) a “reverse modified” sgRNA where only interacting moieties were modified. These gRNAs were co-transfected into HEK293TdEGFP cells with a CMV-SaCas9 plasmid. Cells were processed 48 hours post transfection for TIDE analysis.
Results :
The “loops modified” gRNA performed best with 49.3% EGFP knockdown on TIDE (p<0.0001), with non-significantly lower performances by the “ends-modified” (43.8%, p<0.0001), “heavily modified tail” (34.6%, p = 0.004), “heavily modified body” (32.6%, p < 0.001) and “unmodified” (23.5%, p = 0.01) gRNAs, compared to a non-targeting gRNA “scram” (1.1%). The “heavily modified” (5.0%, p>0.99), and “reverse modified” (1.3%, p>0.99) gRNAs showed no significant editing compared to “scram”.
Conclusions :
Structure-guided chemical modification of SaCas9 gRNA increases editing efficacy, but over-modification in the “heavily modified” gRNA abolished editing. The “heavily modified body” and “heavily modified tail” gRNAs both performed better than the “heavily modified” gRNA, suggesting overall number of modifications is as important as location. The “reverse modified” gRNA shows modification of key gRNA moieties also abolishes editing. Further research is needed to generalise these findings across other species of Cas9 gRNA.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.