CRISPR is seemly turning the table in biological research as once recognized being the bacterial immune system against invading viruses, the CRISPR and its associated system like CRISPR-cas9 with programmable capabilities are now reforming diverse fields of medical research, biotechnology, and agriculture.
What are CRISPRs?
CRISPR (clustered regularly interspaced short palindromic repeats) are DNA segments present in the genomes of prokaryotic organisms such as archaea and bacteria that code for adaptive immunity in prokaryotes against intrusive genetic elements such as viruses and plasmids.
CRISPR-Cas9 where Cas9 is regarded as a DNA-snipping enzyme, and CRISPR is a group of DNA sequences that tell Cas9 where to snip.
PIONEERS OF CRISPRs:
CRISPRs were discovered in E.coli in 1987 by Yoshizumi Ishino and his team, who were exploring a gene responsible for alkaline phosphatase conversion when they accidentally cloned a unique set of repetitive sequences interspersed with spacer sequences.
In the year 2002, Ruud Jansen coined the term CRISPR formerly called “the repeated sequences of DNA”, they even observed the presence of a specific gene in the neighborhood of CRISPR and named it as CRISPR-associated gene (Cas). CRISPR-Cas works like a molecular scissor, slicing DNA into fragments.
The existence of CRISPR/Cas9 in a prokaryote, according to Eugene Koonin, is proof of previous viral infections. When a bacterium survives a viral attack, it naturally opens up its genome and absorbs the broken fragments of viral DNA as spacers into its nucleic acid, preserving the attacker's genetic ID in its DNA. Bacteria have the ability to transfer their acquired genetic code to future generations quite similar to humans. The genetic code that has been obtained is entirely CRISPR.
Further, in the upcoming year, Jennifer Doudna who is now acknowledged as the mother of CRISPR presented the idea that microbial immunity mechanisms could be harnessed for programmable genome editing and this approach revolutionized the world of genetic engineering as the techniques utilized at that period were tedious and not feasible enough like CRISPR.
CRISPR was co-invented by Emmanuelle Charpentier. Both (Dr. Charpentier and Dr.Doudna) explored guide RNA and Cas9 enzyme-mediated DNA cleavage and its biochemical characterization. Her knowledge of microbiology, biochemistry, and genetics paved the door for the discovery of CRISPR's potential application.
CRISPRs-associated systems: (Cas)
Jansen and group revealed that the CRISPR-associated systems are those clusters of identical genes that accompanied the prokaryote repeat cluster that is CRISPR and this vital information worked as a building block in the development of CRISPR technology.
Researchers discovered critical properties of CRISPR repeat and spacer elements through the computational study of these genomic sequences. During the initial investigation, the CRISPR sequence was found in 40% of sequenced bacteria and 90% of archaea.
Second, CRISPR elements are located near CRISPR-associated (Cas) genes, which are a collection of well-conserved genes.
The collection of non-repeating spacer DNA sequences in the bacteria was perceived as the longings of virus and other genetic elements, that turned the table for the ongoing research.
Cas9 works by unwinding foreign DNA and looking for locations that are complementary to the guide RNA’s 20 base pair spacer region.
How do CRISPRs Work?
The Cas9 which is regarded as RNA guided DNA endonuclease enzyme linked with CRISPR was discovered in the adaptive immunological system of Streptococcus pyogenes.
- pyogenes uses CRISPR to memorize and Cas9 to subsequently interrogate and break foreign DNA, such as invading bacteriophage DNA or plasmid DNA, paving the door for genome editing.
It is regarded as the RNA-guided gene-editing tool that utilizes protein (Cas9) produced by bacteria along with a synthetic guide RNA to institute a double-strand break at a determined site within the genome.
Cas9 works by unwinding foreign DNA and searching for locations that are complementary to the guide RNA’s 20 base pair spacer region. Cas9 cleaves the DNA substrate if it is complementary to the guide RNA.
Field application of CRISPR technology
In the development of therapies, identifying genes that drive cancer progression and maintenance in genetically tractable animals is critical. CRISPR technology has been widely utilized to modify genes in cancer models, giving a quick and easy genetic approach for identifying and studying cancer genetic drivers. CRISPR can also be used to modify several genes in order to gain a better understanding of the genomic complexity of human cancers.
The production of mammalian cell lines with single (or perhaps several) gene deletions is possible with CRISPR, allowing for far faster pharmacological testing of targeted medicines.
- In animal models, CRISPR has been used to establish cancer therapy. CRISPR systems have the benefit of being able to edit the genome of somatic cells to introduce driver mutations, eliminating the need for time-consuming germline cell manipulation.
Cancer immunotherapy, or the process of generating or potentiating an antitumor immune response, is becoming a popular treatment option for a variety of tumors. High specificity and effectiveness of the immune system make the utilization of CRISPR appealing for cancer treatment. CRISPR systems are being used to increase immunotherapy efficacy by increasing potency, lowering toxicity and manufacturing costs, and simplifying the development of new immunotherapeutic techniques.
CRISPR has turned out to be the best-known genome editing tool than any other in terms of editing efficiency. As it is easier and far more feasible to use due to its reprogramming ability which can be utilized with the addition of a short guide RNA.
The ability to diagnose the disease quickly and accurately is a basic requirement for effective treatment and the mitigation of long-term consequences. CRISPR technology has the ability to amplify the DNA and RNA even if present in trace amounts which enable highly specific detection and via this disease based on the nucleic acid-based biomarkers can be utilized for diagnostic purpose. CRISPR systems are an essential component of a microbial adaptive immune system that distinguishes foreign nucleic acids based on their sequence and then degrades them using endonuclease activity linked with the CRISPR-associated (Cas) enzyme. The initial CRISPR-based diagnostic approaches relied solely on Cas9 variations that identify double-stranded DNA (dsDNA).
- In CRISPR-based diagnostic, the quantification can be done in the range of picomolar to micromolar in which CRISPR-based collateral cleavage activity correlates with target concentration.
- The CRISPR technology’s applicability is considerably more incredibly diverse than what is described here. In theory, advances in CRISPR biology and biotechnology will continue to tremendously facilitate CRISPR gene therapy research and other CRISPR-based applications in the upcoming decades.
As said by Nobel laureate Sydney Brenner, “Progress in research depends on new techniques, new discoveries, and new ideas, presumably in that order.” CRISPR-based technologies have unquestionably raised interest with an unprecedented toolset. CRISPR-Cas9 will be hailed as one of the primary tools that enabled breakthrough discoveries and practical scientific development. CRISPR applications have already broadened our understanding of genome control and architecture in living cells in a variety of biological kingdoms.
CRISPR technology is opening doors for a better future and has the ability to revolutionize multiple areas like molecular biology, medicine, and biotechnology with its effectiveness.