ACS nanoJul 18, 2026

Designing Guide DNA for Accurate RNA Targeting by Cas12a

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jul 19, 2026

Abstract

Cas12a can achieve up to 21-fold enhanced sequence selectivity using a split guide DNA design.

Cas12a can switch its function from targeting DNA to targeting RNA through engineered guide DNA sequences.
The split guide DNA design concept allows for greater sensitivity to nucleotide variations, aiding in precise targeting.
Cas12a's activation is influenced by specific regions of the guide DNA, with a focus on the scaffold-proximal area.
This approach enables single-nucleotide discrimination among closely related RNA sequences, such as those in the let-7 family.

Simplified

Cas12a is highly accommodative toward noncanonical activation pathways to the extent of flipping its identity to be a DNA-guided RNA-targeting effector. A sequence engineering approach was used to systematically identify desirable guide DNA (gDNA) sequence motifs to achieve comparable RNA targeting efficiency as the canonical RNA-guided Cas12a with good selectivity down to single-nucleotide mismatch. Importantly, we introduced a split gDNA design concept with greater energetic differences arising from subtle nucleotide changes to probe the key spacer features for effective Cas12a-gDNA activation. Similar to the canonical RNA-guided activation pathway, Cas12a was found to engage actively in the "seed-like" scaffold-proximal region while the scaffold-distal region was largely hybridization-driven. We further evolved the split gDNA design to enhance the sequence selectivity by up to 21-fold compared to a single gDNA design and achieve single-nucleotide discrimination among representative let-7 family members. This study has established a gDNA sequence design framework to reprogram Cas12a as a precise RNA targeting platform.

Full Text

Full text is available at the source.

View Full Text ↗

Designing Guide DNA for Accurate RNA Targeting by Cas12a

Abstract

Full Text

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