Cas12based

Cas12based refers to systems that use CRISPR-associated endonuclease Cas12 and its variants (notably Cas12a, Cas12b, Cas12c) to perform programmable DNA targeting. Cas12 proteins are single-effector enzymes from Class 2, Type V CRISPR systems that employ a CRISPR RNA guide to recognize a DNA sequence adjacent to a protospacer-adjacent motif (PAM) and introduce double-strand breaks. Compared with Cas9, Cas12 family members often require different PAMs and tend to generate staggered cuts. Cas12a, for example, recognizes a T-rich PAM and produces cuts distal to the PAM, while Cas12b and other subtypes differ in PAM preferences and temperature optima. In many Cas12 enzymes, binding to the target also triggers non-specific cleavage of single-stranded DNA, a phenomenon known as collateral or trans-cleavage activity.

This collateral activity underpins diagnostic approaches that detect nucleic acids with high sensitivity. Cas12-crRNA complexes are combined with reporter molecules; upon target recognition, collateral cleavage releases signals that indicate the presence of the target sequence. Cas12-based diagnostics have been developed for various viral and bacterial pathogens and have been adapted for multiplex detection.

Cas12-based methods are also applied to genome editing and functional genomics. Editing with Cas12 typically uses guide RNAs and cellular DNA repair pathways to achieve knockouts, insertions, or precise edits, with donor templates guiding homologous recombination. Engineering efforts aim to broaden PAM compatibility, improve specificity, and create variants for base editing or transcriptional regulation when fused to effector domains. Applications span agriculture, biotechnology, and medical diagnostics, while challenges include off-target effects, delivery, regulatory considerations, and ethical implications.