CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) along with Cas proteins form the CRISPR-Cas immune system in archaea and bacteria. The bacterial CRISPR system has been adapted to edit genomes in a wide variety of species. The CRISPR-Cas cleaves DNA at a specific sequence allowing the modification of the gene.
CRISPR in Prokaryotes
The CRISPR method is used by bacteria to protect themselves from infections by viruses. When a virus attacks a bacteria, the bacteria detects its presense and produces two types of short RNA. One of the RNA's contains the same sequence as that of the viral DNA. The two RNA's then form a complex with a protein called Cas9. Cas9 is a nuclease that cuts the viral DNA leading to loss of viral function.
There are three main steps to the CRISPR/Cas system in Prokaryotes. The first step, Insertion, allows Cas1 and Cas2 to detect the foreign DNA, cleave a protospacer, and attach it to the target DNA. The next step, crRNA processing, can be carried out through 3 unique mechanisms, although the primary pathway is Type II. In Type II, the sequence is transcribed and cleaved by Cas9 and RNase III. In the final step, Interference, additional trimming occurs at the 5’ end and mature crRNAs are created. The target sequences then interact with the PAM sequence before the invading DNA is degraded.
For eukaryotes, the TypeII pathway is hijacked. After the DNA is degraded, there are two repair mechanisms that can be carried out - NHEJ (non-homologous end joining) and HDR (homology directed repair). In NHEJ, there is a double stranded break caused by Cas9 which is glued or ligated by DNA ligase IV present at the deletion site. On the other hand, in HDR, the proteins recruited will try to search for complementary bases of DNA because the missing bases will be present on the template strand. Recombination then occurs wherein the broken strand bends into the template and then the DNA polymerase will resynthesize the cut. The only problems in these are that NHEJ is very error prone and HDR is very slow.
- ↑ https://www.ncbi.nlm.nih.gov/pubmed/25574773
- ↑ B Alberts, A Johnson, J Lewis, D Morgan, M Raff, K Roberts et al. (2015) Molecular Biology of the cell Sixth edition, page 497-498, Garland Science, New York, USA
- ↑ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3384332/
- ↑ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3079308/