11.12: CRISPR and crRNAs
Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different stages to attack a reinfecting virus. In the acquisition stage, the protospacer region of viral DNA is cleaved by CRISPR systems. The specific protospacer region is identified for cleavage with the help of a protospacer adjacent motif (PAM) present in the target viral DNA. The cleaved protospacer sequence is then incorporated into the bacterial CRISPR locus. In the expression stage, the CRISPR and CAS genes are transcribed to produce pre-CRISPR RNA (crRNA) and the Cas mRNA. The pre-crRNA is then processed to produce mature crRNA. In the interference stage, crRNA and the translated Cas protein form a ribonucleoprotein complex that targets and cleaves the viral DNA in a sequence-specific manner.
CRISPR-Cas systems can be divided into three distinct types characterized by their Cas protein types. In Type I systems, Cas3 has helicase as well as nuclease activity. Multiple additional Cas proteins create a double-stranded break in viral DNA. In Type II systems, the nuclease Cas9 acts alone to cleave the DNA. In addition to crRNA, Type II systems also have trans-activating CRISPR RNA (tracrRNA) which is required for the maturation of the crRNA. In Type III systems, Cas10 has an unknown function, but like the type I system, it needs multiple proteins for the DNA cleavage. The type III system can also target RNA for cleavage. Type I and Type III are found in both bacteria and archaea, while to date type II has been only found in bacteria. Compared to conventional genome editing techniques like restriction enzymes, the CRISPR-Cas system is simpler to use can target multiple genes in the same experiment.; therefore, it has emerged as a powerful genetic engineering tool and is widely being used to modify the genome of both prokaryotic and eukaryotic organisms.