dCas9

dCas9, or dead Cas9, is a catalytically inactivated variant of the Cas9 endonuclease derived from bacteria such as Streptococcus pyogenes. Mutations in the nuclease domains, typically D10A in the RuvC domain and H840A in the HNH domain, abolish DNA cleavage while preserving single-guide RNA-directed DNA binding. As a result, dCas9 serves as a programmable DNA-binding scaffold rather than a nuclease.

In CRISPR interference (CRISPRi), dCas9 blocks transcription by occupying promoter or coding regions, repressing gene expression. In CRISPR activation (CRISPRa), dCas9 is fused to transcriptional activators (examples: VP64, p65, Rta; composite systems like dCas9-VPR) to increase transcription of target genes. dCas9 can also be fused to chromatin-modifying enzymes to edit epigenetic marks, or to fluorescent proteins for live-cell imaging of genomic loci. It is used with guide RNAs of about 20 nucleotides to direct the complex to specific DNA sequences adjacent to a PAM, typically NGG for SpCas9.

Limitations and considerations include that binding depends on the presence of a PAM, and off-target binding can occur. Specificity improvements rely on careful guide design, high-fidelity Cas9 variants, or truncated gRNAs. Because dCas9 does not introduce double-strand breaks, unintended recruitment of cofactors remains a consideration. Delivery methods include plasmids, mRNA, or ribonucleoprotein complexes; efficiency and expression levels influence outcomes. Applications span basic research, functional genomics, and therapeutic design, though clinical use remains experimental and subject to regulatory oversight.