dCas12a

Dead Cas12a (dCas12a) refers to catalytically inactivated variants of the CRISPR-Cas12a (Cpf1) nuclease. Like active Cas12a, dCas12a binds target DNA in a sequence-specific manner guided by a CRISPR RNA (crRNA) and requires a Cas12a-compatible PAM, typically TTTV, for binding. Mutations disable the nuclease activity, so dCas12a can bind without introducing double-strand breaks.

Because it does not cut DNA, dCas12a is used as a programmable DNA-binding platform for gene regulation, epigenetic editing, and imaging. In transcriptional regulation, dCas12a is fused to transcriptional activators or repressors to upregulate or downregulate target genes in bacteria and eukaryotic cells. The ability of Cas12a to process CRISPR RNA arrays can enable multiplex targeting, allowing simultaneous regulation of multiple loci using a single transcript, though practical use depends on whether the RNase activity is retained in the specific dCas12a variant.

Cas12a-based tools differ from Cas9-based ones: Cas12a uses a single crRNA without a tracrRNA, recognizes a T-rich PAM on the 5' side of the target, and generally generates staggered cuts in nuclease-active form. In the dead form, binding suffices for regulatory outcomes rather than cleavage.

Researchers also deploy dCas12a for imaging, by fusing fluorescent proteins to track genomic loci, and for epigenetic editing by tethering chromatin modifiers to modulate local chromatin states. Multiple Cas12a variants have been engineered to broaden PAM compatibility or improve specificity.