How does CRISPR gene editing work?

CRISPR gene editing is a two-part system. A Cas enzyme, most commonly Cas9, cuts DNA, and a short piece of guide RNA tells the enzyme where to cut. After the cut, the cell tries to repair the break, and scientists use that repair process to change, remove or add DNA letters. [1] [2]

The first component is Cas9, which acts as a pair of molecular scissors that cuts both strands of DNA. The second is the guide RNA, which is about 20 bases of pre-designed sequence sitting within a longer RNA scaffold. The scaffold binds to the Cas9 protein, and the designed sequence guides Cas9 to the right part of the genome. [3]

The guide RNA is made to be complementary to the target DNA, meaning its bases match the target sequence. Because RNA and DNA bind when their sequences match, the guide scans the DNA for its target. One educational source compares this to the Find function on a computer, with the guide RNA as the word being searched for. In theory, the guide binds only to the intended target and nowhere else in the genome. [4] [5] [3]

The Wellcome Sanger Institute describes five steps. Researchers identify a target sequence and create a guide RNA for it. The guide RNA binds to the target DNA. Cas9 binds to the guide RNA, then cuts both strands of DNA. Finally, the cell's own repair mechanisms fix the cut, introducing a genetic change. [6]

A cell senses broken DNA and tries to fix it, and gene editing takes advantage of this. The more common route is nonhomologous end joining, in which the cell directly reattaches the cut ends. This process is not always perfect, and bases can be added or lost at the cut. That often stops the gene from working, which lets scientists study what the gene does. Cutting at two places around a gene can also remove it. [5] [2]

The second route is homology directed repair. Researchers supply a DNA donor template with the new sequence in the middle and longer homology arms on each side that match the DNA around the cut. The cell uses the template as its guide, so the new sequence is copied into the repair. This allows highly specific edits, even changing a single nucleotide. [5] [2]

The Innovative Genomics Institute notes that editing does not always work perfectly and lists three kinds of problems that scientists look for. Off-target effects occur when the Cas protein cuts in the wrong place, which may be harmless but could impair or kill cells. On-target rearrangements occur when the cut is in the right place but repair produces the wrong edit. Immunogenicity is the possibility that a CRISPR-based tool provokes a dangerous immune response in an organism. [1]

Cas9 is the most common and best-studied enzyme, but other CRISPR tools use different Cas enzymes, such as Cas3 or Cas10. [5] [1]