CRISPR is a gene-editing system, that is equivalent to scissors, for editing genes. It advances as a prominent tool in the field of gene editing. It employs the cutting-edge CRISPR-Cas 9 technology that earned Emmanuelle Charpentier and Jennifer .A. Doudna the Nobel Prize in Chemistry in 2020. It came about through a preliminary research project, which has been aiming at discovering how bacteria fights viral infections. This technology allows scientists to make changes to the DNA in the cells that could allow us to cure genetic diseases. Thus starts the quantum leap of technology that may have far-reaching relevance in microbiology, biomedicine, and agriculture.

What is CRISPR?

CRISPR is the acronym for CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS. It naturally occurs in bacteria and archaea as a defence system against invading viruses. When the virus attacks the bacteria, the bacteria incorporate pieces of the virus's DNA into their genomes. These segments of the virus and the genetic pattern between them are the CRISPR. Now, the bacteria utilize the CRISPR segments as a template to create strengths of RNA whenever the virus attacks again. The CRISPR RNA carries along-with it a protein, i.e. CAS9, to the target location on the viral DNA and disables the virus by cutting its DNA. This artificial guide RNA binds Cas9, and they both act on the target DNA, thereby inducing some conformational changes and makes it active. This new gene-editing technology has the potential to revolutionize genetic engineering.

Source: cdn.the-scientist.com

History

The idea of CRISPR-Cas9 started way back in 1987, even though the term "CRISPR-Cas 9" was published for the first time in March 2002. However, it was published as an aspect of genomic engineering in 2012 by Jennifer .A. Doudna and Emmanuelle Charpentier. Ms. Emmanuelle Charpentier started studying the entire mechanism in an organism called 'Streptococcus pyogenes' which is a bacterium that can cause very severe infections such as pharyngitis, tonsillitis, and scarlet fever. This bacterium called 'Streptococcus pyogenes' has a CRISPR system and consists of a single gene, encoding a protein called 'Cas9' that has been genetically modified, to be required for the functioning of CRISPR. This study laid the foundation for the visualization of this magnificent field of Gene-editing and therapy. It marked the stepping stone to a revolution in the history of biology.

Gene-editing

Scientists have attempted to utilize this cutting-edge research mechanism to make precise cuts or edits in any DNA. Gene-editing technique is, basically, inserting, removing, or modifying DNA in the genome by using Nucleases or Molecular Scissors, which is been used in CRISPR Cas9. With CRISPR Cas9 multiple genes can be targeted simultaneously, which is referred to as Genome-Editing. Cas9 enzymes along-with CRISPR sequences form the basis of technology known as CRISPR-Cas9 that can be used to edit genes within organisms. This complex machinery recognizes DNA, goes, and cuts DNA at a specific location by Cas enzyme which is “CRISPR-associated-protein 9”, thereby catalyzing the cutting of DNA.

Mechanism

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CRISPR-Cas prevents bacteriophage infection, conjugation, and natural transformation by degrading foreign nucleic acid that enters the cells. This system provides immunity to bacteria which then undergoes a natural process when the microbe is invaded by a bacteria fudge. The story starts with a bacterial immune system, which depicts how a bacteria fights viral infection. The three main steps to acquire immunity in bacteria are adaptation, RNA biogenesis, and interference. Cas9 protein searches for target DNA by binding with sequences that match its protospacer adjacent motif (PAM) sequence. This protein is responsible for locating and cleaving target DNA. A customized RNA molecule called the “guide RNA” along with Cas9 protein can latch onto the target DNA sequence, which can then be modified, repaired, or deleted. Guide RNA is engineered to have a 5’ end that is complementary to the target DNA sequence. This artificial guide RNA targets and binds to a specific DNA sequence and gets attached to the Cas9 enzyme, which is basically a nuclease that cuts DNA and forms a Cas9 complex.

Application

Over the years CRISPR Cas9 technique was applied in the treatment of cancer, sickle cell anaemia, and infectious diseases such as TB and Malaria. It has a role in the advancement in the treatment of diseases, investigation, prevention, increased crop yield. CRISPR promises to eradicate HIV, restore vision, create medicines, make better biofuels, and create drought and disease-resistant crops. It’s most recent application was utilized, in the FELUDA test, where the research team at the Institute of Genomics and Integrative Biology used the CRISPR-Cas9 technology to detect the viral genome of SARS-CoV 2, the virus that causes COVID-19. They have designed the guide RNA complementary to target the SARS-CoV 2 genome sequence. The amplification part is essential for increasing the probability of detection of the virus. The mixture in the FELUDA test comprises of:

1. Amplified Viral DNA
2. Guide RNA
3. Cas9 Protein

A nasopharyngeal swab is necessary for the test, which is more comprehensible than the regular RT-PCR (Reverse Transcriptase – Polymerase Chain Reaction). The steps include the extraction of the genetic material (RNA) from the sample, and the subsequent step is a one-step RT-PCR, which involves the conversion of RNA to DNA, and with the help of transcriptase and its amplification to multiple copies. This very process is a single-step procedure. It brings forward a piece of simple machinery with the usage of a shorter time. One more example of a coupled technology is SPRINT (Sherlock-based Profiling of IN Vitro Transcription), which can be used to detect a variety of substances such as Metabolites in patient samples or contaminants in environmental samples. Thereby, it is further been explored for detection and inactivation of the novel coronavirus, "SARS-CoV 2".

It is a potential tool for developing virus-resistant crop variety by developing biotic and abiotic resistant traits. It can also be used to eradicate insect pesticides and herbicide-resistant weeds. In animals, it can bring induced changes in the morphological and genetic level. Gene-editing will reduce the risk of immune responses and rejection during transfusion reactions.

Conclusion

Summing it up, apart from ethical barriers, it may have mosaic problems of safety and efficiency, leading to the wrong attachment. The challenges include specificity, efficacy, and efficient delivery. However, CRISPR-Cas9 is a plank to breakthrough technology, that has various advantages in the field of medicine and purportedly holds the promise for treating more complex diseases such as cancer, heart diseases, mental illnesses, human immunodeficiency virus (HIV) infection, eliminate some other diseases, and also solve problems related to world hunger, pandemic, etc., thereby giving a ray of hope to billions of people providing a faster and a precise way of editing genes. CRISPR-Cas9 immune system has led to a revolution in genome-editing and engineering technologies.

References

• Question and answers about CRISPR, broadinstitute.org
• James C Hu, Four ways this revolutionary gene editing tool to change the world, nbcnews.com
• Niranjana Lakshmi, Explain how does India feluda COVID-19 test work?, science.thewire.in
• CRISPR, Wikipedia.org
• A Nobel Prize for genetic scissors, (2020), Nature Materials.
• Ruud Jansen, Jan D.A. van Embden, Wim. Gaastra, Leo M Schouls, (2002) Identification of genes that are associated with DNA repeats in prokaryotes. Molecular Microbiology.
• Xu et al., (2019) CRISPR-Edited Stem Cells in a Patient with HIV and Acute Lymphocytic Leukemia, N Engl J Med.