11.11:
CRISPR and crRNAs
Bacteria continuously face infections from bacteriophages, viruses that infect bacteria.
To deal with this threat, bacteria have evolved a sophisticated adaptive immunity system known as the CRISPR-Cas system to destroy bacteriophage DNA, in case of reinfection.
This system involves three different processes – incorporation of the bacteriophage DNA segment into the bacterial genome, production of the CRISPR RNA and the Cas protein, and CRISPR RNA-Cas mediated cleavage of the bacteriophage DNA.
When a bacteriophage attacks, it attaches to the surface of the bacterial cell and inserts its DNA into the bacteria which is then cleaved by the bacterial system.
A short segment of the bacteriophage DNA is then added into specific regions of the bacterial genome, called CRISPR, clustered regularly interspaced short palindromic repeats. These are genomic regions where bacterial sequence repeats are interspersed with the short varying spacer sequences from different bacteriophages.
These spacer sequences serve as a memory for the bacteria of all the bacteriophages that have previously attacked. Bacteria use spacer sequences to rapidly identify and destroy DNA from a particular type of bacteriophage if it attacks again.
The transcripts from the CRISPR region are processed to produce CRISPR RNA molecules, around 30 nucleotides long, that contain the spacer sequence and the nearby bacterial repeat sequence.
The CRISPR-associated or Cas systems encode the Cas protein. This Cas protein then associates with the CRISPR RNA molecule to form a ribonucleoprotein complex.
When the same type of bacteriophage attacks again, its DNA is recognized by its specific spacer sequence present in the CRISPR RNA. Associated Cas protein then, either alone or with the help of multiple proteins, cuts both strands of the bacteriophage DNA.
The principles of the CRISPR-Cas system and its components can be used to knockdown or modify any gene in an organism using its complementary guide RNA.
Different types of the CRISPR-Cas system are present in bacteria and archaea. Among them, the CRISPR-Cas9 system is one of the most well studied and widely used for different practical applications, as it can be easily and effectively reprogrammed to target different genes.
11.11:
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.
Suggested Reading
- Thurtle‐Schmidt, Deborah M., and Te‐Wen Lo. "Molecular biology at the cutting edge: a review on CRISPR/CAS9 gene editing for undergraduates." Biochemistry and Molecular Biology Education 46, no. 2 (2018): 195-205.
- Rath, Devashish, Lina Amlinger, Archana Rath, and Magnus Lundgren. "The CRISPR-Cas immune system: biology, mechanisms and applications." Biochimie 117 (2015): 119-128.
- Wang, Haifeng, Marie La Russa, and Lei S. Qi. "CRISPR/Cas9 in genome editing and beyond." Annual Review of Biochemistry 85 (2016): 227-264.
- Adli, Mazhar. "The CRISPR tool kit for genome editing and beyond." Nature Communications 9, no. 1 (2018): 1-13.