SNiP-ing Success with CRISPR

HideRNAs. Dead-RNAs.¹ CRISPR GUARD RNA.² 

…all names for a special kind of guideRNA (gRNA). While gRNAs ‘guide’ Cas9 to genomic sites to induce double-strand breaks, hideRNAs, dead-RNA and CRISPR GUARD RNA ‘hide’ these sites from being cut. Their protective function is conferred by simply being shorter than standard gRNA. Rather than having spacer sequences that are 17-24 nt, hideRNAs have spacer sequences that are 8-15 nt in length. Interestingly, Cas9 that is complexed with these trimmed gRNA can bind but not cut targeted sites.³

Harmsen et al. reasoned that hideRNAs with perfect base complementarity to the edited site could protect the edited site from re-cutting by Cas9. Re-cutting is especially common when gene editing does not significantly change the targeted site, as is the case with single base edits. Indeed, Harmsen et al. show that co-transfection of gRNA with hideRNA significantly improves efficiency of single-base gene editing in vitro and in vivo.

Read on if you think your CRISPR experiments are plagued by re-cutting, which can decrease gene-editing efficiency and introduce undesired mutations. Co-transfection with hideRNA may be a quick fix!

Citation

Title: HideRNAs protect against CRISPR‑Cas9 re‑cutting after successful single base‑pair gene editing
Authors: Tim Harmsen, Colin Pritchard et al.
Journal: Scientific Reports, Volume 12, 10 June 2022.
DOI: 10.1038/s41598-022-13688-y
Product Usage: CRISPR gene-editing experiments in mouse embryonic stem cells (IB10) and HEK 293FT cells were performed with TransIT®-LT1 Transfection Reagent. A 120-nt ssODN template was co-transfected with a single plasmid that encoded all of the CRISPR gene-editing components (i.e. gRNA, Cas9, puromycin resistance gene, hideRNA).

Discover more ways transfection is applied in CRISPR-mediated gene editing by visiting our Citations Database.