Originally identified as acquired immune system in some bacteria and archea against genetic attacks, CRISPR/Cas-9 system has now been established as modern gene editing tool that is capable of acting against large number of diseases caused by genetic molecules (1, 2). Apart from applications in gene therapy, CRISPR/Cas system has also been used variably, from protein production to crop improvement, for its capability to insert double stranded break at specific sites increasing the probability of occurrence of homological recombination (3, 4). The guided endonuclease activity of this system enabled knocking down of specific unwanted genes (5). In addition, modification of the Cas protein also allowed the system for enhanced worth (6).
Figure: CRISPR/CAS – 9 systems causing double stranded breaks in DNA. a) Natural CRISPR/CAS-9 system b) Modified CRISPR/CAS-9 system c) Sequences for guide RNA and tracrRNA-crRNA hybrid. (Adapted from: Sander and Joung, 2014).
When invading mobile genetic molecules try to overcome the bacterial host genome equipped with CRISPR/Cas-9 system, some sequences from those foreign invaders are incorporated into the CRISPR locus. From these modified sequences, CRISPR RNA, which has sequences complimentary to the part of original invader – also called protospacer, transcripts are produced. Along with the protospacer sequence, these CRISPR RNA, also termed as guide RNA, sequences also harbour repeat sequences that hybridize with another RNA molecule called trans activating crRNA (tracrRNA) forming a complex. This complex of hybridised nucleic acid molecules is capable of recruiting the Cas-9 protein, an endonuclease enzyme, activating the immune system. Thus activated CRISPR/Cas-9 system is capable of scanning the host genome for the presence of foreign sequences complimentary to protospacer part of the crRNA. The enzyme Cas-9 is also capable of inserting double stranded breaks at these specific site in the invader, which are adjacent to protospacer adjacent motifs – PAM (short sequences – usually ‘NGG’) (7). After the cleavage of specific sites in the DNA, it will be repaired by non-homologous end joining mechanism that is highly prone in causing insertion and deletion mutating the original gene (8).
It is the flexibility of crRNA molecules and Cas-9 enzyme to be transformed easily that has made this system a revolutionary tool for gene or genome editing. After the advent of next generation sequencing tools and with the advancements in technologies for synthesizing artificial nucleic acids, CRISPR/Cas-9 system has become a next generation tool for genetically manipulating the diversity of living organisms.
- Chylinski K, Le Rhun A, Charpentier E. The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biology. 2013;10(5):726-37.
- Guo S, Lv Y, Lin Y, Lin K, Peng P, Wu Y, et al. CRISPR/Cas9 Systems: The Next Generation Gene Targeted Editing Tool. Proc Natl Acad Sci India Sect B – Biol Sci. 2015;85(2):377-87.
- Xie K, Yang Y. RNA-Guided genome editing in plants using a CRISPR-Cas system. Mol Plant. 2013;6(6):1975-83.
- Roointan A, Morowvat MH. Road to the future of systems biotechnology: CRISPR-Cas-mediated metabolic engineering for recombinant protein production. Biotechnology and Genetic Engineering Reviews. 2017;32(1-2):74-91.
- Barrangou R, Birmingham A, Wiemann S, Beijersbergen RL, Hornung V, Smith AVB. Advances in CRISPR-Cas9 genome engineering: Lessons learned from RNA interference. Nucleic Acids Res. 2015;43(7):3407-19.
- Oakes BL, Nadler DC, Savage DF. Protein engineering of Cas9 for enhanced function. Methods in Enzymology: Academic Press Inc.; 2014. p. 491-511.
- Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014;32(4):347-50.
- Wyman C, Kanaar R. DNA double-strand break repair: All’s well that ends well. In: Campell, Anderson, Jones, editors. Annual Review of Genetics2006. p. 363-83.