The Cas9 protein is a central component of the CRISPR-Cas9 gene-editing technology. Often described as “molecular scissors,” this protein can be programmed to cut specific DNA sequences. Cas9 originated in bacteria, where it functions as part of an adaptive immune system to defend against invading viruses. By incorporating pieces of viral DNA into their genome, bacteria produce guide molecules that direct Cas9 to recognize and neutralize future infections.
The Two Major Lobes of Cas9
The Cas9 protein has a bilobed structure, organized into two major sections: the Recognition (REC) Lobe and the Nuclease (NUC) Lobe. The REC lobe is responsible for binding to the guide RNA (gRNA), the molecule that provides the target coordinates within the genome.
The NUC lobe contains the protein’s catalytic activity, meaning it cuts the target DNA. This structure can be visualized as a tool with a handle and a cutting head. The REC lobe acts as the handle by holding the guide molecule, while the NUC lobe is the cutting head that performs the DNA cleavage. This division of labor allows for a coordinated, multi-step process. The two lobes are connected by a bridge helix that assists in guide RNA recognition.
The Recognition (REC) Lobe Domains
The primary function of the REC Lobe is to bind the guide RNA (gRNA) molecule. This gRNA contains a sequence of about 20 nucleotides that directs the Cas9 protein to a complementary sequence in the target DNA. The REC lobe is composed of smaller domains, REC1 and REC2, which work together to hold the gRNA in the correct orientation. This interaction is a key step in the gene-editing process.
The binding of gRNA to the REC lobe induces a conformational, or shape, change in the Cas9 protein. This change activates the enzyme, shifting it into a state ready to search for and bind to DNA. The gRNA and a portion of the target DNA strand are held in the space between the REC and NUC lobes.
The Nuclease (NUC) Lobe Domains
The NUC lobe is where the DNA-cleaving action of Cas9 occurs, executed by several domains. The first part of this lobe to engage the target is the PAM-Interacting (PI) domain, which scans DNA for a short sequence known as the Protospacer Adjacent Motif (PAM). The widely used Cas9 protein from Streptococcus pyogenes recognizes the PAM sequence “NGG”. Cas9 will not cut the DNA unless the PI domain first finds this sequence adjacent to the target site.
Once the PI domain confirms the PAM sequence, the DNA double helix unwinds, allowing the gRNA to check for a match with the target DNA strand. If the sequences are complementary, two other domains in the NUC lobe are activated. The HNH domain cleaves the target strand, which is the DNA strand complementary to the guide RNA.
Simultaneously, the RuvC domain cleaves the non-target strand. The coordinated cuts from the HNH and RuvC domains result in a double-strand break in the DNA. This break initiates the cell’s natural repair processes, which can be harnessed to make specific edits to the genetic code. The HNH domain is generally large and mobile, while the RuvC domain is more static.
Engineering Cas9 Through Domain Modification
Understanding Cas9’s domains allows scientists to engineer the protein for functions beyond DNA cutting. By modifying or deactivating specific domains, researchers have developed new molecular tools. These variants use the targeting ability of Cas9 while altering its enzymatic activity to achieve different outcomes at a specific location in the genome.
One common modification is “dead” Cas9, or dCas9, where mutations deactivate the HNH and RuvC nuclease domains, eliminating its ability to cut DNA. Guided by gRNA, dCas9 can still bind to a specific DNA sequence. This binding can physically block other proteins from accessing the DNA, silencing a gene in a process called CRISPR interference (CRISPRi). Alternatively, functional proteins can be fused to dCas9 to activate gene expression, a technique known as CRISPR activation (CRISPRa).
Another variant is the Cas9 nickase, created by deactivating only one of the two nuclease domains (either HNH or RuvC). This results in an enzyme that cuts only a single strand of DNA, creating a “nick” instead of a double-strand break. This approach allows for more precise editing strategies that reduce the risk of errors associated with double-strand break repair. The nicks can prompt the cell’s repair machinery to make specific changes to the DNA.
Building on these modifications, scientists developed advanced tools like base and prime editors. These editors fuse a Cas9 nickase or dCas9 to another enzyme, such as a deaminase or a reverse transcriptase. The Cas9 component homes in on the correct DNA target, while the attached enzyme performs a direct chemical modification of the DNA bases. This allows for single-letter changes in the genetic code without inducing a double-strand break, offering high precision for gene correction.