Cluster regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated (Cas9) system identified as heritable adaptive immunity regulator or provider to prokaryotes against foreign genetic elements and invaders.
Introduction
The CRISPR which is a locus in genomics of prokaryotes that is bacteria and archaea. The CRISPR locus was identified in 1987 containing 5 highly homologous repeats of 29 nucleotide separated by 30 nucleotide basis in E.coli(1). Whenever a foreign genetic element in the form of virus(bacteriophages, plasmids and transposons) enter into the bacteria or prokaryotes the bacteria chop the small part of DNA and incorporates into CRISPR region on its own(2,3). In a subsequent invasion of bacteria by same virus results into quick action of bacteria by CRISPR-Cas9 nucleases guided by CRISPR RNA and transactivating RNA, results in double strand break in DNA of invaders.The identification of this system has revolutionize not only medical field but otherbiological system. TheCRISPR-Cas technology is being applied in many disciplines including agriculture, medicine, basic sciences, fuel generation,pharmacology and genetic engineering(4). The CRISPR-Cas system has been recently unfold as a flexible tool for biological and medical research. In this system a single guide RNA (sgRNA) governs endonuclease Cas9 to a targeted DNA for site specific changes. After revelation of CRISPR French scientist proposed its role in cell immunity against phage infection and degrade DNA. CRISPR genome editing allows to rapidlyproduce cell and animal model to speedup research of a particular disease.
Role of CRISPR in diagnostics
CRISPR-Cas9-based gene editing has been used to diagnose various pathogens.It can detect pathogenic DNA. CRISPR-Cas9 sgRNAhas helped to understand the mechanism as howa-hemolysin, a Staphylococcus aureus virulence factor causescytotoxicity(5). The Cas13 can detect cancer cell line. It produces signal via reporter attached to the RNA and mutagenesis can be detected through this signaling.Human pappiloma virus can be detected by CRISPR.Cas13 based specific high-sensitivity enzymatic reporter unlocking(SHERLOCK) program can reveal Zika virus (ZIKA) and dengue virus at concentration as low as 1 copy per microliterin urine or serum. If CRISPR finds ZIKA virusthenCas cuts the virus and cut the molecule of RNA holding a reporter (labeled) which helps in diagnosis. If virus is not present in sample the reporter remains intact. The CRISPR CAS-13 targets RNA rather DNA and chews up other RNA that is related to target (collateral cleavage). This system has high sensitivity and can detect drug resistance.CRISPR-Cas9 with optical DNA mapping can recognize antibiotic resistant gene(6). It can amplify RNA and thus can help diagnosis of diseases caused by RNA viruses. It has been reported thatnucleic acid amplification can be done by CRISPR. Polymerase converts DNA into RNA AND Cas13detects specific sequences in the RNA. Cas13 allow to prepare sample without nucleic acid extraction. Screening and diagnostic can be done in case of pregnancy to diagnose the DOWNsyndrome. It not only detect virus but also detect its resistance and other phenotypes. It can differentiate between ZIKA virus and Dengue virus in the same sample although clinically it shows similarities. However different strains of ZIKA virus and Dengue virus can also detected via SHERLOCK. Highly pathogenic viruses that evolved globally like Ebola virus and Middle East Respiratory Syndrome (MERS) and new strain of corona virus can be detected via CRISPR technology. By using CRISPR-Cas13a H7N9 can be detected quickly. This technology has also successfully been employed to diagnose parasitic diseases including Toxoplasmosis and Chagas disease (7).
Role of CRISPR in therapy
First implementation could be in cancer research. Using CRISPR the removed T-cells are altered to extract gene that encodes for a protein called PD-1. Some tumors are able to bind with this protein on the surface of immune cell and command them not to attack. Modified cells are then reimplanted with higher capacity to attack cancer cells. The CRISPR has been used to extract PD-1 as well as change molecule on surface of immune cell to find and attack tumor. The CRISPR/Cas9 system is used to edit human immunodeficiency virus (HIV-1) genome and block its expression. It also remove internal viral gene from host cell chromosome. By CRISPR technology bone marrow stem cellscollection is done to make them produce fetal hemoglobin in blood disorderthat binds oxygen much finer than adult form. In case of hemophilia CRISPR therapy play a role where gene editing tool is carried directly to liver.Blindness can be treated by using CRISPR therapy in which function of light sensitive cells are rehabilitate before sight is completely lost. In vivo genome editing leads to reduce intra ocular pressure (IOP). Gene therapy target mutated gene in case of congenital blindness. In Huntington’s disease CRISPR play role in treatment by cutting abnormal genes in DNA sequence. In case of corona virus, treatment role of CRISPR technology is not well understood but it is speculated that it can help to get rid of this virus from the body of infected patient. In case of cystic fibrosis the macrophages that are functioning abnormally can be switched to normal through CRISPR technology. The CRISPR is revolutionary technology that is altering the genesis of next generation products. By CRISPR we can recognize, cut, cleave and replace mutant genes with healthier one. The CRISPR-Cas can help the immune system to drive, enhance and generate better fat in dairy culture to make better yoghurt, helpful for gut bacterial population. Human genetic modification can be done in the earliest stage of embryonic development that passed on to future generation. If parents want to see blue eyed baby it can be done through the use of CRISPR by changing in OCA2 gene. Gene editing can also be done in fertilized eggs allowing creation of transgenic animals with targeted mutations (8). Modification in germ line cells can be transferred to that individual as well as its descendants.
CONCLUSIONS:
CRISPR has a bright future in the diagnosis and treatment, with fewer error. The CRISPR is a cheaper, quicker and can diagnose the disease earlier and has ahuge advantage in treating the cancer and other diseases.
REFERENCES:
- Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A (1987). 2. Garneau JE, Dupuis ME,Villion M,Romero DA (2010). 3. Deltcheva E, Chylinski K, Sharma CM, Pirzada ZA(2011). 4. Mali P,Yang L,Esvelt KM,Church GM(2013). 5. Winter SV, ZychlinskyA, Bardoel BW(2016). 6. Muller V, Rajer F, westerlund F, Sandegren L, Nyberg LK(2016). 7. Sidik SM, Hackett CG, Tran F, Westwood NJ, Lourido S (2014). 8. Dana V. Foss, Megan L. Hochstrasser(2019).
Authos: M. Tariq Javed*, Amna Kanwal, Afsheen Fazil, M. Atif Raza Department of Pathology, Faculty of Veterinary Science, University of Agriculture Faisalabad.