BY: SREELAKSHMI (MSIWM012)

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), are the hallmark of a bacterial defines system that forms the basis for CRISPR-Cas9 genome editing technology. In the field of genome engineering, the term “CRISPR” or “CRISPR-Cas9” is often used  to refer to the various CRISPR-Cas9 and -CPF1 and other systems that can be edited  to target specific stretches of genetic code and to edit DNA at precise locations and also  for other purposes, such as for new diagnostic tools. With these systems, researchers can permanently modify genes in living cells and organisms. In the future, it may be possible to correct mutations at precise locations in the human genome in order to treat genetic causes of disease. Other systems now available includes systems such as CRISPR-Cas13 which has the ability to target RNA and provide alternate ways for use and with unique characteristics that have been leveraged for sensitive diagnostic tools, such as SHERLOCK. Francis Mojica who is a microbiologist discovered Crisper.In2017 gene editing experiment on human embryo for the correctness of heart condition was successful. It was done to edit a gene MYBPC3 which caused hypertrophic cardiomyopathy(HCM),which affects 1 in 500.It has no cure and it causes cardiac arrest which leads to sudden death.

HISTORY OF CRISPRs

CRISPRs were first discovered in archaic and later in bacteria’s by Francisco Mojica, a scientist at the University of Alicante in Spain. He stated that CRISPRs serve as part of the bacterial immune system, defending against invading viruses. It consists of repeating sequences of genetic code which was interrupted using spacer sequences which are the remnants of genetic code from past invaders. Mojica’s theory was experimentally demonstrated in 2007 by a team of scientists led by Philippe Horvath. In January 2013, the Zhang lab published the first method to engineer CRISPR to edit the genome in mouse and human cells. Later many of the scientists and teams contributed to the understanding and development of the CRISPR system from the initial discovery to the first demonstrations of CRISPR-mediated genome editing. Feng Zhang and his team have trained thousands of researchers in the use of CRISPR genome editing technology and by sharing more than 40,000 CRISPR components with academic laboratories around the world.

 WORKING OF CRISPR

CRISPR mainly uses spacer sequences which are transcribed into short RNA sequences capable of guiding the system to matching sequences of DNA. When the target DNA is found, Cas9 which is one of the enzymes produced by the CRISPR system binds to the DNA and cuts it, shutting the targeted gene off. Modified versions of Cas9 is used by the researchers which can activate gene expression instead of cutting the DNA. These techniques allow researchers to study the gene’s function.

Research also tells that CRISPR-Cas9 can be used to target and modify typos in the three-billion-letter sequence of the human genome which can be helpful in treating genetic disease.

 IMPORTANCE OF CRISPR-Cas9 COMPARED TO OTHER GENOME EDITING TOOLS

CRISPR-Cas9 is proved to be an and efficient and tailored alternative to other existing genome editing tools. CRISPR-Cas9 system itself is capable of cutting DNA strands. They do not need to be paired with separate cleaving enzymes as other genome editing tools need. They can also be easily matched with tailored “guide” RNA sequences designed to lead them to their DNA targets. They are generally called as gRNA. Thousands of such gRNA sequences are already created and are available to the research community for studies. CRISPR-Cas9 can also be used to target multiple genes simultaneously, which is another advantage while comparing with other gene-editing tools that.

 DIFFERENCE BETWEEN CRISPR-Cpf1 AND CRISPR-Cas9

CRISPR-Cpf1 differs drastically from CRISPR-Cas9 while looking through the angle of research.Cas9 which is a DNA cutting enzyme in its natural form forms a complex which consist of two RNAs which are required for cutting activity. The Cpf1 is a simple system that   requires only a single RNA. The Cpf1 enzyme is a smaller than the standard SpCas9 which makes it easy to deliver into cells and tissues.Cpf1 cuts the DNA in different manner than Cas9.  The Cas9 complex cuts the DNA into both strands at the same place by leaving the blunt ends that often undergoes mutations while they are rejoining. Whereas Cpf1 complex cuts in the two strands which are offset and leaves short overhangs near the exposed ends. This is done to get help with precise insertion which allows the researchers to integrate a piece of DNA more efficiently and accurately.Cpf1 cuts far away from the recognition site which indicates that even if the targeted gene becomes mutated at the cut site it can still be cut again which allows multiple opportunities for correct editing to be done. The Cpf1 system provides flexibility in choosing the target sites. Similar to Cas9, Cpf1 complex must first get attached to a short sequence known as a PAM, and targets must be chosen which are adjacent to naturally occurring PAM sequences. The Cpf1 complex recognizes very different PAM sequences than those of Cas9. This could be an advantage while targeting, for example the malaria parasite genome and even the human genome.

 Scientific uses of CRISPR 

CRISPR genome editing allows the scientists to quickly create cell and animal models, which are used by researchers to accelerate their research into diseases such as cancer and mental illness. In addition, CRISPR is now being developed as a rapid diagnostic method.

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