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Introduction
The technology of CRISPR offers a precise and facile mechanism revolving around the molecular editing of cells, tissue, and whole organisms. Moreover, CRISPR technology has enabled to alleviate the genetic disorders in animals and cancer therapies, which is why it is considered a potent tool for genome manipulation in plants, animals, and microorganisms. In the arena of research, the CRISPR edited genome has wide application as controlling transcription, conducting genome-wide screens, modifying epigenomes, and imaging chromosomes. Beyond biomedical applications now this technology is expediting crops and livestock breeding as well as engineering novel antibiotics by controlling the gene drives of diseases. hence the purpose of Seattle Genova is to review the current and future applications of CRISPR which can be utilized in vitro as well as in vivo and applied in research fields of therapeutics, xenotransplantation, and other industrial microbes.
Engineering CRISPR Systems
The CRISPR genome editing experiments are utilized to exploit the host cell machinery to repair the targeted genome precisely at the particular site of CAS-9. The possibility of mutation arises either through non-homologous end joining (NHEJ) or homology-directed repair of the targeted genome (HDR) (Müller et al.,2014). Through the NHEJ small insertion or deletion, the genome produces whereas HDR utilizes a native DNA template to replace the targeted allele with an alternative sequence by recombination (Slaymaker et al., 2016).
Genome-Wide Screens
The technology of CAS-9 has harnessed genome-wide studies which provide large-scale DNA and RNA synthesis through improvements in the lentiviral library generation and propagation (Housden et al.,2015). Moreover, the application of CRISPR-CAS9 technology, it had provided the studies of non-coding sequences and characterize enhancer regulatory sequences. Furthermore, the CRISPR-CAS9 technology screening of various exploiting libraries comprises thousands of sgRNA to identify the tumor-related and metastasis genes among them (Valletta et al.,2015).
Animal Models
CRISPR technology can engineer large animal models to analyze the mechanism of immune rejection and transmission of diseases across the species barrier. Moreover, CRISPR-mediated genome editing potentially expedites the development of large animal models of human diseases which includes primates (Yang et al., 2015). Therefore, CRISPR accelerates suitable therapies for identification and mimics human physiology.
Cell Therapy Applications
The therapeutic use of CRISPR technology has paved paths for many cell therapy applications. Various research has been conducted on CRISPR-based genome editing which was successful in editing the defective genotypes in vitro. In that case, the editing of cystic fibrosis transmembrane regulator sequence inside iPSCs cells has produced altered or corrected versions of cells that differentiate into mature airways epithelial cells (Firth et al.,2015). Moreover, the CRISPR-Cas9 approaches also influence the disease alleles of muscular dystrophy and Fanconi anemia and set the pace for clinical implementations (Nelson et al., 2016).
Another milestone achieved through CRISPR technology is its implementation in vivo model organisms as it has been implemented in the adult mouse liver for alteration of fumarylacetoacetate hydrolase (Fah)-related genes and improving the muscle function in cardiocytes, muscle stem cells, myofibrils, and live animals. Most recently, autosomal dominant retinitis pigmentosa through the rhodopsin mutation was altered through the injection of plasmids encoding CRISPR–Cas9 (Guan et al., 2018).
Antimicrobial and Antiviral Applications
The CRISPR-Cas system has multiple antimicrobial and antiviral applications by maintaining homeostasis in cells and tissues through the multiple robust DNA repair pathways, as bacterial DNA possess only a few primitive DNA repair process. This feature of CRISPR technology has paved the path for bacterial programmed death (Gomaa et al.,2014). The engineering of a self-targeting CRISPR-Cas system which is associated with the nucleases and cleaves the DNA and is programmed to target any bacterial species. Thus, the sequence-specific antibiotics generated could selectively modulate the bacterial populations and eliminate the pathogens (Sternberg et al., 2015).
Agriculture Application
In the field of agriculture, CRISPR-based genome engineering would improve classic breeding as in cows, chickens, and pigs. The animal breeders are already prone to the trait-associated chromosomal markers to selectively advance the valuable traits and could be improved through the utilization of CRISPR technology, which utilizes to protect against viruses. Additionally, CRISPR is also used to engineer the production of medical products or tissues as was observed in pigs through the knock-in of human albumin cDNA into the pig Alb locus and enhancing the production of albumin (Peng et al.,2015). Moreover, CRISPR-enabled engineering also plays a vital role in commercial and model crops to increase yield and growth as well as improve drought tolerance and breed crops with improvised nutritional properties (Ricroch and Hénard-Damave, 2016). CRISPR-based gene targeting was also harnessed to combat the plant pathogens as has been observed in the case of tomato yellow leaf curl virus in Nicotiana Benthamian (Ali et al., 2015).
Food Industry
The application system of CRISPR in the field of bacterial studies has been widely studied and contains multiple paths, which include vaccinations, genotyping, engineering probiotic cultures, and controlling the uptake and dissemination of antibiotic-resistance genes by bacteria (Selle & Barrangou 2015). Moreover, the commercial success of the CRISPR-Cas immune system to vaccinate the Streptococcus thermophilus starter cultures have been utilized in dairy fermentation and paved many ways for the technology of CRISPR in the food industry (Garneau et al., 2010). In addition to that, the beneficial bacteria which are immunized have been taken against the dissemination of genes that encode antibiotic resistance and engineer industrial bacteria, fungi, and yeast to manufacture green chemicals, biofuels, and biomaterials. Recent research portrays the importance of CRISPR-mediated vaccination which could be processed as molecular recording events, by capturing the synthetic DNA sequences which are useful for data storage inside bacterial, and possibly other genomes.
Biological Control Applications
The availability of CRISPR technology has created many genes that drive Cas9 which acquire the trait and Cas9 machinery, further coupled enabling the rapid trait propagation inside a population. For example, the gene utilized in Anopheles gambiae (vector for malaria) was to drive a recessive female sterility genotype with 90% transmission to progeny rates (DiCarlo et al., 2015). Hence, this approach has an important role in suppressing the spread of malarial disease among the human species (Hammond et al., 2016). Similar is the case with anti-Plasmodium falciparum CRISPR systems and controlling the spread of disease agents and successful implementation on a broad scale.
Refrences
Ali, Z., Abul-Faraj, A., Li, L., Ghosh, N., Piatek, M., Mahjoub, A., ... & Mahfouz, M. M. (2015). Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. Molecular plant, 8(8), 1288-1291.
DiCarlo, J. E., Chavez, A., Dietz, S. L., Esvelt, K. M., & Church, G. M. (2015). Safeguarding CRISPR-Cas9 gene drives in yeast. Nature biotechnology, 33(12), 1250-1255.
Firth, A. L., Menon, T., Parker, G. S., Qualls, S. J., Lewis, B. M., Ke, E., ... & Verma, I. M. (2015). Functional gene correction for cystic fibrosis in lung epithelial cells generated from patient iPSCs. Cell reports, 12(9), 1385-1390.
Garneau, J. E., Dupuis, M. È., Villion, M., Romero, D. A., Barrangou, R., Boyaval, P., ... & Moineau, S. (2010). The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 468(7320), 67-71.
Gomaa, A. A., Klumpe, H. E., Luo, M. L., Selle, K., Barrangou, R., & Beisel, C. L. (2014). Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. MBio, 5(1), e00928-13.
Guan, X., Luo, Z., & Sun, W. (2018). A peptide delivery system sneaks CRISPR into cells. Journal of Biological Chemistry, 293(44), 17306-17307.
Hammond, A., Galizi, R., Kyrou, K., Simoni, A., Siniscalchi, C., Katsanos, D., ... & Nolan, T. (2016). A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nature biotechnology, 34(1), 78-83.
Housden, B. E., Valvezan, A. J., Kelley, C., Sopko, R., Hu, Y., Roesel, C., ... & Perrimon, N. (2015). Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Science signaling, 8(393), rs9-rs9.
Müller, Maximilian, Ciaran M. Lee, Giedrius Gasiunas, Timothy H. Davis, Thomas J. Cradick, Virginijus Siksnys, Gang Bao, Toni Cathomen, and Claudio Mussolino. "Streptococcus thermophilus CRISPR-Cas9 systems enable specific editing of the human genome." Molecular Therapy 24, no. 3 (2016): 636-644.
Nelson, C. E., Hakim, C. H., Ousterout, D. G., Thakore, P. I., Moreb, E. A., Rivera, R. M. C., ... & Gersbach, C. A. (2016). In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science, 351(6271), 403-407.
Peng, J., Wang, Y., Jiang, J., Zhou, X., Song, L., Wang, L., ... & Zhang, P. (2015). Production of human albumin in pigs through CRISPR/Cas9-mediated knockin of human cDNA into swine albumin locus in the zygotes. Scientific reports, 5(1), 1-6.
Ricroch, A. E., & Hénard-Damave, M. C. (2016). Next biotech plants: new traits, crops, developers and technologies for addressing global challenges. Critical reviews in biotechnology, 36(4), 675-690.
Selle, K., & Barrangou, R. (2015). Harnessing CRISPR–Cas systems for bacterial genome editing. Trends in microbiology, 23(4), 225-232.
Slaymaker, I. M., Gao, L., Zetsche, B., Scott, D. A., Yan, W. X., & Zhang, F. (2016). Rationally engineered Cas9 nucleases with improved specificity. Science, 351(6268), 84-88.
Sternberg, S. H., LaFrance, B., Kaplan, M., & Doudna, J. A. (2015). Conformational control of DNA target cleavage by CRISPR–Cas9. Nature, 527(7576), 110-113.
Valletta, S., Dolatshad, H., Bartenstein, M., Yip, B. H., Bello, E., Gordon, S., ... & Boultwood, J. (2015). ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts. Oncotarget, 6(42), 44061.
Yang, L., Güell, M., Niu, D., George, H., Lesha, E., Grishin, D., ... & Church, G. (2015). Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science, 350(6264), 1101-1104.
