The term CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) refers to a naturally occurring genetic system that has been repurposed into a tool for editing the genomes of living organisms. This technology allows scientists to make highly specific modifications to an organism’s DNA. The system pairs a protein, known as Cas (CRISPR-associated), with a guide molecule that directs the protein to a desired location in the genetic code. Understanding the native structure of the CRISPR locus and the mechanism of its engineered components is key to understanding this technology.
The Natural Role of the CRISPR Locus in Bacterial Immunity
The CRISPR locus is a segment of DNA found within the genomes of many bacteria and archaea, where it functions as an adaptive immune system. This system provides prokaryotic cells with a defense mechanism against invasive genetic elements, primarily from viruses called bacteriophages and foreign plasmids. The locus features a series of short, identical DNA sequences, known as repeats, separated by unique segments of non-repetitive DNA called spacers.
These unique spacer sequences are genomic remnants of past infections. When a bacteriophage invades a bacterial cell, the Cas proteins capture a small fragment of the viral DNA, known as a protospacer, and insert it into the CRISPR locus as a new spacer.
If the cell is infected again by the same virus, the CRISPR locus is transcribed into a long RNA molecule, which is then processed into small CRISPR RNAs (crRNAs), each containing one of the spacer sequences. These crRNAs then associate with Cas proteins to form a surveillance complex that patrols the cell, searching for matching foreign DNA. This provides sequence-specific immunity.
Engineering the Core Components for Gene Editing
Scientists simplified the naturally complex bacterial immune system into a two-part system optimized for genome editing. This engineered tool consists of the Cas9 enzyme and a synthetic single guide RNA (sgRNA). The Cas9 protein acts as the molecular scissor, capable of cutting both strands of the DNA double helix.
The single guide RNA is the component that makes the system programmable, directing the Cas9 enzyme to a specific genomic location. The sgRNA is a chimeric molecule. This sgRNA contains a short sequence of approximately 20 bases that is complementary to the target DNA sequence, ensuring highly specific binding.
By changing the 20-base sequence of the sgRNA, researchers can reprogram the Cas9 enzyme to target any gene. Once the Cas9 enzyme is bound to the sgRNA, the resulting ribonucleoprotein complex is introduced into a cell and begins scanning the genome for a match.
The Molecular Mechanism of Targeting and Repair
The Cas9-sgRNA complex searches the cell’s nucleus for potential target sites in the DNA. For the Cas9 enzyme to bind successfully, it must first recognize a short DNA sequence called the Protospacer Adjacent Motif (PAM), which is located immediately downstream of the target site. The PAM sequence ensures the complex is positioned correctly to begin the editing process.
Upon locating the PAM, the Cas9 enzyme unwinds the double-stranded DNA, and the sgRNA attempts to pair its bases with the complementary target sequence. If a match is confirmed, the Cas9 enzyme undergoes a conformational change that activates its nuclease domains, leading to the creation of a double-strand break (DSB) in the DNA molecule, typically a few base pairs upstream of the PAM. The cell attempts to repair this break.
The outcome depends on which of the cell’s two major DNA repair pathways is utilized. The most common pathway is Non-Homologous End Joining (NHEJ), which ligates the two broken DNA ends back together. This process is error-prone, often resulting in small random insertions or deletions at the break site. These changes disrupt the gene’s reading frame and effectively inactivate or “knock out” the gene’s function.
The second pathway is Homology-Directed Repair (HDR), which requires a template DNA sequence to guide the repair. Scientists can provide an engineered donor DNA template alongside the Cas9 system, allowing the cell to use this template to accurately repair the double-strand break. HDR is used to insert a new sequence, correct a specific mutation, or replace a segment of DNA.