CRISPR: A New Era

 

 What Is CRISPR?
CRISPR technology is a simple yet effective method for genome editing. It enables scientists to readily change DNA sequences and gene function. Correcting genetic abnormalities, curing and controlling the spread of diseases, and boosting crops are just a few of its many potential applications. However, its promise also raises ethical concerns.
"CRISPR" (pronounced "crisper") is a popular abbreviation for "CRISPR-Cas9." "CRISPR" stands for "clusters of regularly interspaced short palindromic repeats."

Discovery of CRISPRs
CRISPRs are DNA sequences that repeat themselves in the genomes of prokaryotes like bacteria and archaea. CRISPRs were discovered in E. coli in 1987 by Yoshizumi Ishino and his team, who were investigating a gene for alkaline phosphatase conversion when they accidently cloned a unique set of repetitive sequences interspersed with spacer sequences. [Ishino, 2018] The purpose of these arrays, however, remained a mystery due to a lack of sufficient DNA sequence data.  

In 1993, researchers led by J.D. van Embden in the Netherlands discovered that different strains of Mycobacterium tuberculosis had different spacer sequences between the DNA repeats. They characterized M. tuberculosis strains based on their spacer sequences, a  technique known as spoligotyping. [Sola, 2015] Subsequently, these sequences were
identified in several other bacterial and archaeal genomes. Researchers Francisco Mojica and Ruud Jansen were the first to refer to them as CRISPRs [Morange, 2015].

How does it work?
The CRISPR-Cas9 system is made up of two essential molecules that cause a mutation in
the DNA:

• The enzyme Cas9 is one of them. This functions as a pair of 'molecular scissors,' allowing portions of DNA to be inserted or removed by cutting the two strands of DNA at a precise point in the genome.

• A strand of RNA known as guide RNA (gRNA). This is made up of a short pre-designed RNA sequence (about 20 bases long) embedded in a larger RNA scaffold. The scaffold binds to DNA, and the pre-programmed sequence directs Cas9 to the correct location in the genome. This ensures that the Cas9 enzyme cuts the genome at the correct location.     

 The guide RNA is intended to locate and bind to a certain DNA sequence. The RNA bases in the guide RNA are complementary to those in the genome's target DNA sequence. This means that the guide RNA will only bind to the target sequence and not to other parts of the genome, at least in theory. Cas9 follows the guide RNA to the same spot in the DNA  sequence as the guide RNA and cuts through both strands of DNA. The cell recognizes that the DNA has been damaged and attempts to repair it at this point. Scientists can manipulate the DNA repair system to make alterations to one or more genes in a cell's genome [What Is CRISPR-Cas9?, 2021].

Three main categories of genetic edits can be performed with\ CRISPR/Cas9:

1. DISRUPT= If a single cut is made, a process called non-homologous end joining can result in the addition or deletion of base pairs, disrupting the original DNA sequence and causing gene inactivation
2. DELETE=A larger fragment of DNA can be deleted by using two guide RNAs that target separate sites. After cleavage at each site, non homologous end joining unites the separate ends, deleting the intervening sequence.
3. CORRECT OR INSERT=Adding a DNA template alongside the CRISPR/Cas9 machinery allows the cell to correct a gene, or even insert a new gene, using a process called homology directed repair. [CRISPR/Cas9, 2020]. 

CRISPR-Cas Systems as an Adaptive Immune Response
CRISPR systems were assumed to be a unique DNA repair mechanism in thermophilic archaea and bacteria when they were initially found. (What Is CRISPR-Cas9?, 2021) Mojica and colleagues discovered that the spacer sequences were comparable to those found in bacteriophages, viruses, and plasmids in the early 2000s. They determined that viruses are  unable to infect bacteria that have homologous spacer sequences, implying that these sequences are involved in prokaryotes' adaptive immune system. [Ishino, 2018] When a virus infects a prokaryote, the spacer sequences in CRISPR arrays are translated into short CRISPR RNA (crRNA), which directs the CRISPR-associated sequence (Cas) protein to cleave complementary DNA or RNA viral sequences, depending on the kind of CRISPR-Cas system. CRISPR-Cas systems work as a defensive mechanism to prevent the same virus from infecting cells again.

Applications of CRISPR

• CRISPR-Cas9 offers a lot of promise as a technique for treating a variety of genetically based medical diseases, such as cancer, hepatitis B, and even high cholesterol. 
• Many of the proposed uses involve altering the genomes of somatic cells, but the possibility to modify germline cells has sparked considerable interest and discussion.
• CRISPR systems are already being used to alleviate genetic disorders in animals and are likely to be employed soon in the clinic to treat human diseases of the eye and blood. 

The Future of CRISPR-Cas9: Beyond Genome Editing
Researchers have tweaked the Cas9 nuclease to perform targeted epigenome editing since the discovery of the CRISPR-Cas9 gene-editing system. Enzymatically dead Cas9 (dCas9) is a modified Cas9 that can be connected to one of numerous epigenome-changing enzymes, such as DNA demethylases, methylases, or acetyltransferases. dCas9, like unmodified Cas9, is guided to the desired genomic region by a guide RNA. The dCas9-enzyme complex, instead of cutting DNA, alters the epigenome at the location. Epigenome editing has the ability to either activate or suppress transcription. Demethylating DNA with enzymes like dCas9-Tet1 or altering histones with dCas9 connected to the histone acetyltransferase p300 enzyme can stimulate transcription. Transcription can be inhibited by
utilising DNA methyltransferase to methylate DNA. Linking dCas9 to enzymes that recruit corepressor proteins can also silence genes.
It is still early days for CRISPR-Cas9 technology. As more applications are uncovered, the sky is the limit!  

References
1. Ishino, Y., et al. History of CRISPR-Cas from encounter with a mysterious repeated sequence to genome editing technology. Journal of Bacteriology, 200, 7 (2018). e00580-17. doi: 10.1128/JB.00580-17.
2. Sola, C., et al. High-throughput CRISPR typing of Mycobacterium tuberculosis complex and Salmonella enterica serotype Typhimurium. Methods in Molecular Biology, 1311 (2015). doi:10.1007/978-1-4939-2687-9_6.
3. Morange, M., et al. What history tells us XXXVII. CRISPR-Cas: The discovery of an immune system in prokaryotes. Journal of Biosciences, 40 (2015). 221–223 https://doi.org/10.1007/s12038-015-9532-6.
4. What is CRISPR-Cas9? (2021, July 21). Yourgenome. https://www.yourgenome.org/facts/what-is-crispr-cas9
5. CRISPR/Cas9. (2020, February 17). CRISPR. http://www.crisprtx.com/gene editing/crispr-cas9
6. Makarova, K.S. et al. A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis. Nucleic Acids Research, 30, 2 (2002). 482–496. doi: 10.1093/nar/30.2.482. 

By: Batool Murtaza