Nuclease-dead CRISPR-associated protein 9 (dCas9) (dCas9)

Nuclease-dead CRISPR-associated protein 9 (dCas9) is a modified version of the Cas9 enzyme, most commonly derived from Streptococcus pyogenes, in which the catalytic domains (HNH and RuvC) are inactivated by specific mutations such as D10A and H840A [1, 12]. Unlike wild-type Cas9, dCas9 does not cleave DNA but retains its ability to bind to specific genomic loci guided by a single-guide RNA (sgRNA) [1, 9]. This DNA-binding capability allows dCas9 to serve as a versatile scaffold for gene regulation, where it can physically block RNA polymerase to repress transcription (CRISPR interference or CRISPRi) or, when fused to effector domains, activate gene expression (CRISPR activation or CRISPRa) [8, 14, 15]. In a therapeutic context, dCas9-based systems are being explored to treat diverse conditions, including cancer, viral infections, and various genetic disorders, by modulating the expression of endogenous genes without introducing permanent double-strand breaks in the genome [9, 10, 15]. Development of this target also includes the search for small-molecule inhibitors and anti-CRISPR proteins to provide temporal and dose-dependent control over its activity [7, 18]. Safety considerations for dCas9-based therapies focus on potential immunogenicity due to its bacterial origin, delivery challenges, and the risk of off-target binding [3, 12, 18].

Other names

Dead Cas9Catalytically inactive Cas9Nuclease-deficient Cas9dSpCas9Nuclease-dead Cas9

Binds to specific DNA sequences guided by an sgRNA to sterically hinder transcription or recruit transcriptional/epigenetic modulators without inducing double-strand breaks [1, 8, 14].