The Core Components of CRISPR
The CRISPR system, which originated as a bacterial defense mechanism against invading viruses, operates with precision to modify genetic material. At its foundation are two primary components: a specialized enzyme and a guiding RNA molecule. These components work in tandem to locate and act upon specific DNA sequences within a cell.
The enzyme most commonly associated with CRISPR gene editing is Cas9, though other Cas enzymes like Cas12a also exist. Cas9 functions as a molecular scissor, capable of cutting both strands of a DNA molecule. Its role is to execute the genetic modification once directed to the correct location.
The second crucial component is the guide RNA (gRNA), a short RNA sequence designed to bind to the Cas9 enzyme. This gRNA acts like a molecular GPS, steering the Cas9 enzyme to a precise target site in the cell’s genome. The gRNA’s ability to be customized allows researchers to direct Cas9 to virtually any desired DNA sequence.
Targeting the Gene
The gRNA accurately identifies the specific gene segment to be modified. The gRNA contains a unique sequence, typically around 20 nucleotides long, that is complementary to the target DNA sequence in the genome. This complementary pairing ensures that the gRNA precisely binds to the intended genetic location.
For the Cas9 enzyme to successfully bind and cleave the DNA, another short DNA sequence called the Protospacer Adjacent Motif (PAM) must be present immediately next to the target sequence. The PAM sequence is not part of the gRNA but is a crucial signal on the target DNA that Cas9 recognizes. Without the correct PAM sequence positioned adjacent to the target site, Cas9 cannot perform its cutting function.
The specific PAM sequence can vary depending on the type of Cas enzyme used; for the widely utilized Cas9 from Streptococcus pyogenes, the PAM is typically a 5′-NGG-3′ sequence, where ‘N’ can be any nucleotide. This strict PAM requirement enhances specificity, minimizing off-target activity. The Cas9-gRNA complex scans the DNA, unwinding it to check for sites that match both the gRNA sequence and the adjacent PAM.
Making the Precise Cut
Once the guide RNA (gRNA) has successfully led the Cas9 enzyme to the specific target gene, and the Protospacer Adjacent Motif (PAM) has been recognized, Cas9 activates its enzymatic function. Cas9 creates a double-strand break (DSB) in the DNA molecule. This break occurs at the exact location identified by the gRNA, typically three to four nucleotides upstream of the PAM sequence.
This targeted cleavage of both DNA strands is crucial for gene knockout. The cell’s natural response to such a break is to initiate its DNA repair machinery.
The double-strand break signals DNA damage, prompting the cell’s inherent repair pathways. The cell’s subsequent attempt to mend this break is the mechanism that ultimately leads to the gene being rendered non-functional.
The Cell’s Imperfect Repair
After the Cas9 enzyme creates a double-strand break in the target gene, the cell’s natural DNA repair mechanisms are immediately activated. The primary pathway that leads to gene knockout is called Non-Homologous End Joining (NHEJ). NHEJ is an efficient but often error-prone repair mechanism that rejoins the broken DNA ends without the need for a homologous template.
During the NHEJ process, small insertions or deletions, known as indels, frequently occur at the site of the break. These indels, typically 1 to 10 base pairs, result from the cell’s imprecise attempt to ligate the severed DNA strands. The random nature of these insertions or deletions means that there is approximately a two-thirds chance of causing a frameshift mutation within the gene.
A frameshift mutation alters the gene’s reading frame, which is the way the cell reads the genetic code in sets of three nucleotides (codons) to build proteins. When the reading frame is shifted, the downstream codons are incorrectly read, often leading to the premature appearance of a “stop” signal. This premature stop codon results in the production of a truncated, and typically non-functional, protein. By introducing these disruptive indels, the gene is effectively “knocked out,” meaning its intended function is permanently prevented.