An RNA-guided DNA endonuclease complex derived from the adaptive immune system of bacteria and archaea, repurposed into a programmable genome-editing platform for human cells. In 2012, Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer Doudna, and Emmanuelle Charpentier demonstrated that the Cas9 protein from Streptococcus pyogenes can be directed by a chimeric single guide RNA to cleave any target DNA sequence adjacent to a short protospacer-adjacent motif (PAM), followed in early 2013 by Le Cong, Feng Zhang, and colleagues showing multiplex genome engineering in mammalian cells.
Class
Class 2 Type II RNA-guided DNA endonuclease ribonucleoprotein complex
Site-specific double-stranded DNA cleavage 3 base pairs upstream of a 5'-NGG-3' PAM sequence
Clinical / Biotech Use
Exagamglogene autotemcel (Casgevy) for sickle cell disease and β-thalassemia; base and prime editing; CRISPRi/CRISPRa
Structure & Biogenesis
In native Type II bacterial CRISPR systems, invading bacteriophage or plasmid DNA segments (spacers) are integrated into the host CRISPR array, transcribed into precursor CRISPR RNA (pre-crRNA), base-paired with trans-activating CRISPR RNA (tracrRNA), and processed by host RNase III (hydrolase) in the presence of Cas9.
Apo-Cas9 adopts a bilobed architecture comprising an alpha-helical recognition (REC) lobe and a nuclease (NUC) lobe containing two distinct magnesium-dependent catalytic domains (HNH and RuvC) plus a C-terminal PAM-interacting domain; binding the guide RNA rearranges Cas9 into a central DNA-searching channel.
Mechanism of Action
Cas9-sgRNA scans double-stranded genomic DNA in the nucleus by three-dimensional collision until its C-terminal domain recognizes a 5'-NGG-3' PAM on the non-target DNA strand. PAM binding triggers local DNA melting and directional R-loop zipping as the 20-nucleotide spacer of the guide RNA base-pairs with the target DNA strand, beginning at the PAM-proximal 8-to-12-nucleotide seed region. Complete R-loop formation docks the HNH domain to cleave the target DNA strand (complementary to the RNA guide) and the RuvC domain to cleave the displaced non-target DNA strand 3 base pairs upstream of the PAM, generating a blunt double-strand break that cellular DNA repair pathways resolve via non-homologous end joining (NHEJ) or homology-directed repair (HDR).
Biological & Regulatory Roles
Catalytically inactivated "dead" Cas9 (dCas9, carrying D10A and H840A point mutations) retains programmable DNA binding without cleaving, allowing fusion to transcriptional repressors (CRISPRi), activators (CRISPRa), or epigenetic Histone H3 modifiers.
Nickase Cas9 variants (such as D10A Cas9n) fused to cytidine or adenosine deaminases perform base editing (converting single base pairs without double-strand breaks), while fusion to an engineered reverse transcriptase paired with a prime editing guide RNA (pegRNA) enables prime editing.
Interactions & Complexes
Double-strand breaks introduced by wild-type Cas9 activate the p53–p21 checkpoint via the p53/MDM2 DNA-damage response, while HDR relies on S/G2-phase homologous recombination factors including BRCA1 and BRCA2.
Related Class 2 effectors extend the toolkit: Cas12a (Cpf1) recognizes T-rich PAMs to make staggered DNA cuts, whereas Cas13 enzymes target single-stranded mRNA rather than DNA, complementing siRNA and ASO knockdown.
Therapeutic & Biotechnology Applications
In December 2023, exagamglogene autotemcel (Casgevy) became the first FDA-approved therapy to use CRISPR-Cas9, editing the erythroid enhancer of BCL11A in autologous hematopoietic stem cells to reactivate fetal hemoglobin (HBG1 / HBG2) in HBB-mutant sickle cell disease and transfusion-dependent β-thalassemia.
In vivo CRISPR therapies deliver Cas9 mRNA and chemically stabilized sgRNA inside lipid nanoparticles to edit disease genes (such as TTR, KLKB1, PCSK9, and ANGPTL3) directly in the liver.
Pooled lentiviral sgRNA libraries enable genome-wide pooled CRISPR screens to map drug-resistance mechanisms and synthetic-lethal vulnerabilities across human cancer and disease models.