The CRISPR-Cas9 system has revolutionized genetic research by providing a precise and efficient method for genome editing. This technology, adapted from a natural bacterial defense mechanism, allows scientists to make targeted changes to an organism’s DNA. Understanding the mechanics of this system is fundamental to grasping how it functions as a molecular tool.
The Essential Components of Gene Targeting
Gene targeting relies on two main molecular players: the Cas9 enzyme and the guide RNA (gRNA). Cas9 acts as the molecular scissors, a protein known as an endonuclease, capable of cutting DNA. The guide RNA is a short, custom-designed RNA molecule that serves as the navigation system for the enzyme.
The gRNA contains a sequence, typically about 20 bases long, complementary to the target DNA sequence. The Cas9 and gRNA form a complex that scans the cell’s DNA for a matching sequence. The gRNA’s ability to pair with the target DNA directs the Cas9 enzyme to the desired location. The complex binds to the target DNA, positioning Cas9 to cut, but only if a specific DNA sequence is also present immediately adjacent to the target site.
Defining the Protospacer Adjacent Motif
The crucial sequence that acts as a molecular ‘address label’ for the Cas9 complex is the Protospacer Adjacent Motif (PAM). The PAM is a short DNA sequence, usually between two and six base pairs in length, found on the target DNA strand. For the commonly used Cas9 enzyme derived from Streptococcus pyogenes, the canonical PAM sequence is 5′-NGG-3′, where ‘N’ can be any nucleotide base.
The PAM sequence is not part of the protospacer targeted by the guide RNA. This motif is necessary for Cas9 to bind to the DNA and become activated to cut. The Cas9 complex first scans the genome for this specific sequence. Only upon recognizing and binding to a correctly oriented PAM does it begin to unwind the adjacent DNA helix to check for a match with the guide RNA.
Precision Cutting: Locating the Cleavage Site
The specific location of the double-strand break (DSB) made by the Cas9 enzyme is precisely determined relative to the PAM sequence. Once the Cas9-gRNA complex is bound to the target DNA and the gRNA has hybridized to the protospacer sequence, the Cas9 enzyme is activated to cut both strands of the DNA helix. The cleavage occurs within the protospacer region, which is the sequence complementary to the guide RNA.
The exact point of the cut is consistently located three to four base pairs upstream (in the 5′ direction) from the Protospacer Adjacent Motif. For the widely used S. pyogenes Cas9, which recognizes the NGG PAM, the enzyme makes a cut three base pairs away from the start of that NGG sequence. This consistent distance of the cut from the PAM is critical for targeted action.
Cas9 has two distinct nuclease domains, HNH and RuvC, which are responsible for cleaving the two separate DNA strands. The HNH domain cuts the strand complementary to the guide RNA (the target strand) precisely at this position three base pairs upstream of the PAM. The RuvC domain cuts the non-target strand, which contains the PAM sequence, at the same location. This synchronized cutting action typically results in a blunt-ended double-strand break.
The Result of the Cut: DNA Repair Pathways
The double-strand break created by the Cas9 enzyme is immediately recognized by the cell as damage that requires repair. The cell possesses two main pathways to deal with this damage, and the choice between them determines the outcome of the gene editing process. The most common and active pathway in most cell types is Non-Homologous End Joining (NHEJ).
NHEJ is considered an error-prone pathway because it simply ligates the two broken DNA ends back together without using a template. This process often results in the insertion or deletion of a few nucleotides at the repair site, creating small, random changes known as indels. If these indels occur within a gene’s coding region, they can disrupt the gene’s reading frame, effectively turning the gene off.
The second primary mechanism is Homology-Directed Repair (HDR), which is an error-free pathway that uses a homologous DNA sequence as a template for repair. HDR is active mainly during certain phases of the cell cycle, making it less frequent than NHEJ. To achieve precise gene correction or insertion, scientists supply a synthetic DNA template containing the desired sequence change. If the cell uses this supplied template, the break is repaired accurately, allowing for the precise ‘knock-in’ of new genetic information.