Introduction
Through evolution, bacteria and archaea have developed methods to evade and fend off predatory viruses for their survival. One such method is the CRISPR-Cas system of adaptive immunity. The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic material such as those present in plasmids and phage’s and it provides a form of acquired immunity. CRISPR-Cas systems core defining features are their Cas genes and proteins. The CRISPR-Cas 9 system specifically has been used as a genome editing tool since it was originally developed from bacterial genome systems. It can therefore be used to target the genetic material in viruses and be a solution to many viral infections. More specifically the Cas 9 enzyme targets certain sections of the viral genome that prevent the virus from carrying out its normal function (Charpentier, 2015).
CRISPR
CRISPR stands for clustered regularly interspaced short palindromic repeats. They are DNA sequences derived from bacteriophage DNA fragments that had previously infected a prokaryote. CRISPRs use an adaptive immunity system to detect and destroy similar bacteriophagic DNA during future infections. CRISPR loci are composed of alternate repeats and spacers between 18-24 nucleotides long. The CRISPR loci are accompanied by adjacent CRISPR-associated (Cas) proteins that determine the method and steps of immunity (Hille and Charpentier, 2016; Hille et al., 2018; Razzaq and Masood, 2018).
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The Cas 9 protein
The Cas 9 protein is a nucleus protein which can be utilised in gene editing by cutting DNA in specific sites, but its original function lies in bacterial immunity. Cas 9 consists of two parts of RNA: trance activating RNA which is found inside the protein and crRNA which contains a 20-nucleotide sequence. The Cas 9 protein pairs with a guide RNA (gRNA)which in turn guides the enzyme to the desired locus where it unravels and cuts the double-stranded DNA. The Cas 9 enzyme requires a specific PAM (protospacer adjacent motif) sequence located next to where it is supposed to perform the cut. This is a safety system for the bacteria to ensure it does not cut its own DNA. PAM is dependent on which specific CRISPR nuclease you are using. PAM sets limitations to the DNA cutting and this can be combated by choosing a nuclease that fits the targeted sequence or by engineering a nuclease to alter the required PAM sequence (Qi et al., 2013; Zhang, Wen and Guo, 2014). The protein itself could also be mutated for further exploitation for example by making a nick for a single stranded break rather than crossing the whole DNA.
CRISPR-CAS9 mechanism
The CRISPR-CAS mechanism is based on a modified RNA binding to the Cas 9 enzyme as it would to a specific target sequence in the genome. The modified RNA is used to recognize the DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location. Once the DNA is cut, the cell's own DNA repair machinery is used to alter the DNA by replacing an existing segment with a customized DNA sequence (Charpentier, 2015) There are two major pathways of repair. The first one occurs if there are no repair templates and is known as non-homologous end joining. Non-homologous end joining is prone to errors as it consists of joining two DNA strands together. This can easily create mutations such as insertions and deletions but it is also a method to generate knockouts when studying gene function. This means that you would be able to cut the gene you would want to knockout. The other repair system is homology directed repair which is a bit more complicated but can generate very precise modifications and reparations by introducing a DNA template usually through a plasmid which can be double or single stranded. If a transgene has homology to either side of the cut it will insert itself into the specific cut (Figure 1)(Zhang, Wen and Guo, 2014; Hille and Charpentier, 2016; Hille et al., 2018; Razzaq and Masood, 2018).
Drawbacks of the CRISPR-Cas9 system
CRISPR-Cas 9 has great potential in genome editing but unfortunately there are some issues that need to be addressed, such as off-target mutations .Organisms with large genomes often contain multiple DNA sequences that are either identical or highly homologous to the target DNA sequences. CRISPR-Cas 9 cleaves these identical or highly homologous off target DNA sequences as well as the target DNA sequences, and this leads to off-target mutations at these sites. These mutations can lead to numerous undesired effects. Another important factor to off-target mutations is the amount of Cas 9 enzyme expressed in the cell. High concentrations of the enzyme are reported to increase off- site targeting where lowering the concentration of Cas 9 increases specificity and on-target cleavage activity. Therefore, a balance needs to be achieved to maximise its full potential. The second significant issue is CRISPR-Cas 9’s PAM dependency. However, for DNA cleavage this can be optimised by using different Cas 9 orthologs from other bacteria where the PAM sequence is more abundant than in our organism of interest (Hsu, Lander and Zhang, 2014; Zhang, Wen and Guo, 2014).
Potential uses of the CRISPR-Cas 9 system beyond genome editing
A modified CRISPR – Cas 9 system named CRISPRinference (CRISPRi) has been used towards RNA-guided transcription regulation in organisms. It has achieved this by disrupting transcription-related functional sites and therefore taking over the transcription of specific genes. However, this process is irreversible due to permanent DNA modifications (Qi et al., 2013). Gene therapy and its ability to treat many genetic and viral diseases such as Cystic Fibrosis, is a very important use of the CRISPR-Cas 9 system. For Cystic Fibrosis treatment, intestinal stem cells were isolated and developed in culture and the target allele was removed altered and reintroduced into the patient. This diverse system is also used in plants for genetically mutated crops. In Arabidopsis, several genes are targeted, and the mutations are transferred to their offspring successfully (Gratz et al., 2013; Hsu, Lander and Zhang, 2014).
Conclusion
CRISPR-Cas 9 genome editing has the ability to target any sequence. It is a simple, precise, and economical method of gene editing compared to alternative techniques. Its specificity is solely dependent on gRNA and Cas9. It has some off-target effects, but this can be minimized by the production of specific sequences of gRNA. CRISPR-Cas9 has many important purposes such as production of desired, high yield and disease- resistance plants. In humans, it is used for gene therapy and the treatment of genetic and viral diseases. Researchers are still putting the CRISPR-Cas9 system at the heart of many gene-editing related research topics. The CRISPR-Cas9 genome editing technique was a significant contributor to the Nobel Prize in Chemistry in 2020 due to its great advances in the field (Charpentier, 2015).
References
- Charpentier, E. (2015) ‘CRISPR-Cas9: how research on a bacterial RNA-guided mechanism opened new perspectives in biotechnology and biomedicine’, EMBO Molecular Medicine, 7(4), pp. 363–365. doi: 10.15252/emmm.201504847.
- Gratz, S. J. et al. (2013) ‘Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease’, Genetics, 194(4), pp. 1029–1035. doi: 10.1534/genetics.113.152710.
- Hille, F. et al. (2018) ‘The Biology of CRISPR-Cas: Backward and Forward’, Cell, 172(6), pp. 1239–1259. doi: 10.1016/j.cell.2017.11.032.
- Hille, F. and Charpentier, E. (2016) ‘CRISPR-Cas: biology, mechanisms and relevance’, Philosophical Transactions of the Royal Society B: Biological Sciences, 371(1707), p. 20150496. doi: 10.1098/rstb.2015.0496.
- Hsu, P. D., Lander, E. S. and Zhang, F. (2014) ‘Development and Applications of CRISPR-Cas9 for Genome Engineering’, Cell, 157(6), pp. 1262–1278. doi: 10.1016/j.cell.2014.05.010.
- Qi, L. S. et al. (2013) ‘Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression’, Cell, 152(5), pp. 1173–1183. doi: 10.1016/j.cell.2013.02.022.
- Razzaq, A. and Masood, A. (2018) ‘CRISPR/Cas9 System: A Breakthrough in Genome Editing’, Molecular Biology, 07. doi: 10.4172/2168-9547.100021.
- Zhang, F., Wen, Y. and Guo, X. (2014) ‘CRISPR/Cas9 for genome editing: progress, implications and challenges’, Human Molecular Genetics, 23(R1), pp. R40–R46. doi: 10.1093/hmg/ddu125.
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