DNA-guided CRISPR

In native bacterial defense systems, Cas12 activation requires a CRISPR RNA scaffold that forms a pseudoknot structure. Engineered systems replace this with a synthetic single-stranded DNA molecule, referred to as ΨDNA or crDNA.^[4]

Activation requires a Protospacer Adjacent Motif (PAM). The synthetic DNA guide mimics a crRNA framework and includes a 3' or 5' extension that binds the PAM-detecting region of Cas12. This interaction forms a stable deoxyribonucleoprotein (DNP) complex capable of binding complementary RNA strands.^[3] Structural analyses have detailed the molecular basis of this ternary complex.^[5] Target binding is enabled through molecular alignment.

Upon binding to target RNA, the Cas12-DNA complex undergoes a structural shift that activates the collateral cleavage of nearby single-stranded DNA. Depending on the Cas12 variant, the complex can degrade mRNA directly or physically block ribosomes to induce translational repression via no-go decay.^[1] DNA-guided Cas12 systems preferentially bind RNA and rarely interact with single- or double-stranded DNA targets.^[4]

The foundation and system for DNA-guided Cas12 RNA targeting was first established publicly in a November 2024 pre-print by the Jain laboratory.^[6] In 2026, the Jain laboratory and the Hsing laboratory (Wu et al.) published peer-reviewed validations of these guide architectures in Nature Biotechnology.^[1][3] These parallel developments were highlighted in an independent review.^[4] Subsequent optimizations of the guide backbones and spatial positioning enhanced binding affinity across orthologs such as AsCas12a and Cas12i1.

• CRISPR
• CRISPR-Cas12a
• Gene editing
• Synthetic biology