CRISPRdCas9

CRISPRdCas9 refers to a catalytically inactive variant of the CRISPR-associated protein 9 (Cas9) that binds DNA in a guide RNA–dependent manner without cutting. By fusing dCas9 to transcriptional effectors or other functional domains, researchers can modulate gene expression, alter epigenetic marks, or label genomic loci in living cells. The most common form uses mutations in the nuclease domains that abolish cleavage while preserving DNA binding and PAM recognition.

The mechanism relies on dCas9’s retained ability to bind DNA when guided by a single-guide RNA. When positioned at promoters or coding regions, dCas9 can sterically hinder RNA polymerase to repress transcription (CRISPR interference, CRISPRi). Alternatively, dCas9 can be fused to transcriptional activators to upregulate genes (CRISPR activation, CRISPRa) or to enzymes that modify epigenetic marks, such as histone modifiers or DNA methylation enzymes, to alter chromatin state and gene expression.

Applications include transcriptional regulation, epigenome editing, and imaging. CRISPRa and CRISPRi enable tunable gene control without changing DNA sequence. Epigenetic fusions allow targeted modification of methylation or histone marks. dCas9 fused to fluorescent proteins or other reporters enables live-cell imaging of genomic loci.

Limitations and challenges include off-target binding with unintended regulatory effects, variable efficiency depending on genomic context, dependence on PAM sequences near targets, and delivery or expression challenges for in vivo use. Ethical and regulatory considerations accompany uses that regulate gene expression or epigenetic states in humans, distinct from direct genome cleavage.