Background information
The 2020 Nobel Prize in Chemistry has been awarded to Emmanuelle Charpentier and Jennifer A. Doudna who discovered one of sharpest tools in gene editing: the CRISPR/Cas9 genetic scissors. The two female Nobel laureates explained the potentiality of genetic scissors to edit the genes selectively which means this mechanism has enabled geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence. After Emmanuelle Charpentier discovered tracrRNA, a part of the bacterias ancient immune system, CRISPR/Cas 9 on her study of Streptococcus pyogenes, that disarms viruses by cleaving their DNA, she initiated a collaboration with Jennifer Doudna and later their collaborative work succeeded in recreating the bacterias genetic scissors in a test tube (Fernholm & Barnes, 2020). Since its discovery on 2012, it has brought a revolutionary breakthrough on the life sciences and is thought to contribute on curing inherited diseases.
Introduction
Clustered regularly interspaced short palindromic repeats/Crisper associated protein 9, commonly known as CRISPR/Cas9 works like a pair of scissors capable of cutting the genome precisely. The technology consists of a complex composed of a small RNA called guide RNA and the nuclease “Cas9” that binds to a specific DNA sequence complementary to the guide RNA which is then followed by a double strand cut of the DNA by Cas9 (DNA Binding and Cleavage, 2016). DNA repair mechanisms can subsequently be used to introduce precise mutations. In this way, researchers can manipulate DNA to suppress the function of a gene or replace it with a modified gene.
How CRISPER/Cas 9 system works?
In nature CRISPR-Cas9 system is a part of microbial immunity. When a virus infects bacterial cell, the bacteria capture the snippets of viral DNA and use them to create DNA segments known as CRISPR arrays (Genome editing and CRISPR, 2020). The Cas 9 nuclease is directed to its target sequence by gRNA to chop off a piece of viral DNA. The snipped DNA fragment are stored between palindromic CRISPR sequences to retain a genetic memory (Kick et al., 2017) This disables the future infection in defending bacteria from same type of viral attack. After understanding the CRISPR system mechanism in bacteria scientists figured out the ways to reprogram this tool in other species.
CRISPR-Cas 9 system acts in a sequence-specific manner by recognizing and cleaving foreign DNA or RNA. To be functional, this system first requires a CRISPR locus/array containing the hypervariable spacers that the defending host acquires from phages or plasmids, and is located in the host genome in addition to the diverse group of Cas genes that are located in the nearby CRISPR locus which encodes the Cas proteins for the multistep defense against foreign DNA.(Hille & Charpentier, 2016)
The foreign DNA is recognized and it is captured and subsequently integrated as spacers/protospacers derived from phage or plasmid, between the two contiguous repeat sequences located in the CRISPR locus (Singh et al., 2017). Protospacer adjacent motif (PAM) are small nucleotides present near the protospacer which is an important component of this system. The RNA processing transcribes CRISPR locus thereby producing a pre CRISPR RNA (pre-crRNA) and endonucleases cleave the pre-crRNAs into active CRISPR RNAs (crRNAS) or tracrRNA. The crRNA and tracrRNA form a multiprotein complex through the base pairing and with great specificity with the regions of incoming foreign DNA (or RNA). Cas9 enzyme binds DNA in the direction of its corresponding gRNA to PAM sequence (Deveau et al., 2010). The scaffolding ability of tracrRNA along with crRNA specificity can be combined into a single synthetic gRNA (sgRNA) which simplifies guiding of gene alterations to a one component system to increase efficiencies (Guide RNA, 2020). Recognition of the PAM by the Cas9 nuclease is thought to destabilize the adjacent sequence, allowing interrogation of the sequence by the crRNA, and resulting in RNA-DNA pairing when a matching sequence is present. (PAM sequence for Crisper, n.d.)
Usefulness of CRISPR/Cas 9 technology
Genome engineering: CRISPR/Cas9 tool can be applied directly in embryo which reduces the time required to modify target genes compared to gene targeting technologies based on the use of embryonic stem cells. This system helps to identify the most appropriate sequences to design guide RNAs and enables very robust procedures which guarantee successful introduction of the desired mutation.
Tissue regeneration: CRISPR/Cas 9 technology has been used to derive a variety of cells for transplantation such as muscle cells for muscular dystrophy and haemopoietic stem cell for sickle cell anemia.
Live imagining of DNA/RNA: CRISPR/Cas9 technology offer the advantage of tracking dynamic cellular processes of DNA/RNA. Fluorophores can be tagged for proper visualization.
Cancer immunotherapy: New studies have shown that knocking out certain genes causing cancer using CRISPR/Cas 9 tool will be a promising approach for cancer treatment.
Epigenetic editing: This technology can be used without genetically modifying DNA sequence to re-establish normal chromatin structure and correct gene expression.
HIV and Viral diseases: CRISPR/Cas9 can be used in disrupting CCR5 and CXCR4 expression which are potential targets for HIV-1/AIDS gene therapy (Xiao et al., 2019). This technology has been used to inactivate DNA and subsequently RNA viruses in various in vitro, ex vivo and in vivo model systems.
Disease resistant cultivar: CRISPR/Cas9 technology is a highly promising tool for gene editing in crops because of its desirable features like precise specificity, multi gene editing, minimal off-target effects, higher efficiency and simplicity there by degrading the invading pathogenic genes to obtain disease resistant cultivar.
References:
Deveau, H., Garneau, J. E., & Moineau, S. (2010). CRISPR/Cas system and its role in phage-bacteria interactions. Annual Review of Microbiology, 64, 475–493. https://doi.org/10.1146/annurev.micro.112408.134123
DNA Binding and Cleavage. (2016). Tufts. https://sites.tufts.edu/crispr/
Fernholm, A., & Barnes, C. (2020). THE NOBEL PRIZE IN CHEMISTRY 2020 Genetic scissors : a tool for rewriting the code of life. The Nobel Prize, 8. https://www.nobelprize.org/prizes/chemistry/2020/popular-information/
Genome editing and CRISPR. (2020). Medline Plus. https://medlineplus.gov/genetics/understanding/genomicresearch/genomeediting/#:~:text=CRISPR-Cas9 was adapted from,(or closely related ones).
Guide RNA. (2020). Wikipedia. https://en.wikipedia.org/wiki/Guide_RNA#:~:text=Guide RNAs (a.k.a. gRNA%2C sgRNA,process known as RNA editing.&text=For this prokaryotic DNA-editing,to the CRISPR-Cas9 system.
Hille, F., & Charpentier, E. (2016). CRISPR-cas: Biology, mechanisms and relevance. Philosophical Transactions of the Royal Society B: Biological Sciences, 371(1707). https://doi.org/10.1098/rstb.2015.0496
Kick, L., Kirchner, M., & Schneider, S. (2017). CRISPR-Cas9: From a bacterial immune system to genome-edited human cells in clinical trials. Bioengineered, 8(3), 280–286. https://doi.org/10.1080/21655979.2017.1299834
PAM sequence for Crisper. (n.d.). Integrated DNA Technologies. https://www.idtdna.com/pages/support/faqs/what-is-a-pam-sequence-and-where-is-it-located
Singh, V., Braddick, D., & Dhar, P. K. (2017). Exploring the potential of genome editing CRISPR-Cas9 technology. Gene, 599, 1–18. https://doi.org/10.1016/j.gene.2016.11.008
Xiao, Q., Guo, D., & Chen, S. (2019). Application of CRISPR/Cas9-based gene editing in HIV-1/AIDS therapy. Frontiers in Cellular and Infection Microbiology, 9(MAR), 1–15. https://doi.org/10.3389/fcimb.2019.00069
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