dCas

dCas, short for dead Cas, refers to catalytically inactivated CRISPR-associated nucleases, most commonly dead Cas9 (dCas9). These proteins retain sequence-specific DNA binding guided by a single-guide RNA but lack nuclease activity due to mutations in the nuclease domains (for example, D10A and H840A in SpCas9). As a result, they can occupy target genomic loci without cutting DNA, enabling programmable binding and manipulation of the genome.

In operation, dCas proteins act as DNA-binding scaffolds. They can block transcription by sterically hindering RNA polymerase or, when fused to effector domains, recruit transcriptional repressors or activators to modulate gene expression. CRISPR interference (CRISPRi) typically uses dCas9 fused to repressors such as KRAB, while CRISPR activation (CRISPRa) uses activators like VP64, p65, Rta or tripartite fusions such as VPR. Beyond transcriptional control, dCas fusions enable epigenetic editing (for example, targeted histone or DNA methylation changes), chromatin imaging, and the targeted recruitment of chromatin modifiers or DNA repair factors.

Limitations include dependency on PAM sequences, potential off-target binding, and the need for efficient delivery in some systems. While dCas systems do not cut DNA, binding can still influence local chromatin structure. Variants such as dCas12a (dCpf1) offer different PAM requirements and targeting options, broadening applicability and multiplexing potential.

Overall, dCas proteins provide a versatile, programmable platform for studying gene function, regulating gene networks, and labeling genomic loci without introducing DNA breaks.