What is CRISPR-Cas9?
CRISPR-Cas9 is a powerful genome editing tool. With the CRISPR-Cas9 gene editing technique genomes of living organisms may be modified by removing, adding or altering sections of the DNA sequence. CRISPR stands for clustered regularly interspaced short palindromic repeats. Those repeats are found in bacteria’s DNA. They are actually copies of small pieces of viruses.
CRISPR-Cas9 technology was adapted from the natural defense mechanisms of bacteria and archaea. These organisms use CRISPR-derived RNA and various Cas proteins, including Cas9, as an antiviral defence system. They do so primarily by cutting and destroying the DNA of an invader.
When these components are transferred into other, more complex, organisms, it allows for the manipulation of genes, or “editing.”
How does CRISPR-Cas9 work?
The CRISPR-Cas9 system consist of two essential molecules: Cas9 and guide RNA (gRNA).
Cas9 stands for CRISPR associated protein 9. Working like genetic scissors, the Cas9 acts like a pair of molecular scissors, capable of cutting strands of DNA. Cas9 plays a crucial role in the immunological defense of certain bacteria against DNA viruses and plasmids. Although Cas9 is the enzyme that is used most often, other enzymes (for example Cpf1) can also be used.
The gRNA is designed to find and bind to a specific sequence in the genomic DNA. The gRNA has RNA bases that are complementary to those of the target DNA sequence in the genome. The gRNA will only bind to the target sequence. gRNA consists of a small piece of pre-designed RNA sequence (about 20 bases long) located within a longer RNA scaffold. The scaffold part binds to genomic DNA and the pre-designed sequence ‘guides’ Cas9 to the right part of the genome. The gRNA is located right before a common sequence in the genome called Protospacer Adjacent Motif (PAM). This makes sure that the Cas9 enzyme cuts at the right point in the genome.
Bound together, Cas9 and gRNA form a complex. The entire CRISPR-Cas9 complex is brought into the target cells. The Cas9 recognizes and binds to its target sequence in front of the PAM sequence. Activated by the PAM sequence, the Cas9 system then acts as a molecular scissor at its target specific location, leading to a double strand break (DSB). The cell with a DSB created by the Cas9 nuclease will try to repair it in this step, which can happen in two ways. The first option is repairing the break by insertion of a known DNA sequence provided by the scientist. This small DNA sequence can be introduced into the cell by transfection, together with the CRISPR-Cas9 complex. The second option is that the cells natural repair mechanism will join the two ends of the cut DNA together, called non-homologous end joining. This process is of DNA fixing is error-prone as nucleotides can be inserted or deleted by mistake.