Jyoti Arya^1*, Renu Bist²

¹ Department of Zoology, Vedic P.G. College, Jaipur, India.

² Department of Zoology, Centre of Advanced Studies, University of Rajasthan, Jaipur, India. 

*Corresponding Author:Dr. Jyoti Arya, Assistant Professor, Department of Zoology, Vedic P.G. College, University of Rajasthan, Jaipur, India, Tel: 0000-0002-4351-7224; Fax: 0000-0002-4351-7224

Citation: Jyoti Arya, Renu Bist (2023) CRISPR Technology: Harnessing the Power of Gene Editing and Addressing Roadblocks with Cutting-Edge Solutions. Arch Mol Med & Gen 3: 122.

Received: October 02, 2023; Accepted: October 10, 2023; Published: October 20, 2023.

Copyright: © 2023 Jyoti Arya, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)technology has revolutionized the field of genetic engineering, offering unprecedented precision and efficiency in manipulating DNA. In medicine, CRISPR holds great promise for the treatment of genetic diseases. By editing the DNA of affected cells, specific genetic mutations underlying inherited disorders can be corrected. Additionally, CRISPR is being explored as a tool for cancer treatment, where it can enhance the immune system's ability to target and destroy cancer cells. In infectious disease research, CRISPR-based diagnostics and engineering of animal models provide insights into pathogenesis and aid in the development of new therapies. Furthermore, CRISPR shows potential in combating viral infections, including chronic viral diseases like HIV. In agriculture, CRISPR offers significant advancements in crop improvement. By precisely modifying specific genes, crops can be enhanced for disease resistance, environmental stress tolerance, yield potential, and nutritional content. CRISPR enables the development of environmentally friendly pest and disease control strategies, reducing reliance on chemical interventions. In the field of biotechnology, CRISPR has extensive applications. It facilitates drug discovery by elucidating gene function and identifying therapeutic targets. CRISPR is instrumental in functional genomics, unravelling complex biological processes and gene interactions. It drives synthetic biology, enabling the engineering of microorganisms for biofuel production, pharmaceutical synthesis, and industrial processes. It has potential in biomedical research, which is evident through disease modelling, elucidating disease mechanisms, and exploring novel therapies. Moreover, CRISPR plays a role in DNA and genome engineering, allowing precise manipulation of genetic material for diverse applications.

Key Words:CRISPR, Genetic Engineering, Treatment, Biomedical Research, Genetic Diseases.

While CRISPR offers remarkable opportunities, ethical considerations, safety assessments, and regulatory frameworks are crucial to ensure responsible and beneficial use of this technology. By addressing these challenges, CRISPR holds immense potential for transforming medicine, agriculture, and biotechnology, paving the way for innovative solutions to societal challenges. CRISPR is a revolutionary gene editing technology that allows scientists to make precise changes to the DNA of living organisms (Jinek et al., 2012; Gasiunas et al., 2012). It is derived from a natural defense mechanism found in certain bacteria that protects them against viral infections (Mojica et al., 2005).

The CRISPR system consists of two main components: a guide RNA (gRNA) and an enzyme called Cas9. The guide RNA is designed to be complementary to a specific target sequence in the DNA, and the Cas9 enzyme acts as a pair of "molecular scissors" that can cut the DNA at the targeted location.

The process of using CRISPR begins with designing a gRNA that is complementary to the desired target sequence in the DNA. The gRNA is then combined with the Cas9 enzyme and introduced into the cells of the organism. The Cas9 enzyme locates the target sequence in the DNA and makes a cut, creating a double-stranded break in the DNA.

Once the DNA is cut, the cell's natural repair mechanisms come into play. There are two main repair pathways: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ repairs the break by directly joining the cut ends of the DNA, often resulting in small insertions or deletions that can disrupt the gene's function. HDR, on the other hand, can be harnessed to introduce specific changes by providing a DNA template with the desired sequence that can be used to repair the cut.

CRISPR has revolutionized the field of genetic engineering due to its simplicity, efficiency, and versatility. It has been used in a wide range of organisms, including bacteria, plants, animals, and even human cells, opening up possibilities for applications in various fields.

In research, CRISPR has been used to study the function of genes by "knocking out" or disabling specific genes in order to observe the effects (Cong et al., 2013). It has also been used to introduce specific mutations into organisms to study the role of particular genes or to create disease models. CRISPR has the potential for numerous practical applications, including agriculture, medicine, and biotechnology. In agriculture, it can be used to develop crops CRISPR has the potential for numerous practical applications, including agriculture, medicine, and biotechnology. In agriculture, it can be used to develop crops with improved traits, such as increased yield, enhanced nutritional content, and resistance to diseases or environmental conditions. In medicine, CRISPR holds a promise for treating genetic diseases by editing the DNA in affected cells. It could also be used to engineer immune cells to better target and destroy cancer cells. Additionally, CRISPR has potential applications in creating new diagnostic tools and therapies for various diseases. Although conventionally, antibiotics have been used since many years in therapeutics, yet they generate a selective stress in the large intestine on host bacteria, thus leading to multidrug resistance which in turn enhances the growth of resistant bacteria (Mishra et al., 2019).

While CRISPR offers significant benefits, ethical and safety concerns have also been raised. Table 1 summarizes the applications of CRISPR in various disciplines. There are ongoing discussions regarding the potential misuse of this technology, such as creating "designer babies" or making heritable changes that could impact future generations. Additionally, off-target effects, where unintended edits occur in the genome, are a concern that needs to be addressed to ensure the accuracy and safety of CRISPR-based applications. Overall, CRISPR technology has transformed the field of genetic engineering and holds great promise for advancing scientific research and addressing various challenges in agriculture, medicine, and Biotechnology.

CRISPR technology has shown great potential for various applications in medicine. Here are some key areas where CRISPR is being explored:

Gene therapy: CRISPR can be used to correct genetic mutations underlying inherited diseases. By targeting the specific mutation in the DNA, CRISPR can edit the genome of affected cells and potentially restore normal gene function (Xie et al., 2014; Song et al., 2015). This approach has been tested in preclinical and early clinical trials for diseases like sickle cell anaemia, beta-thalassemia, and certain types of inherited blindness.

Cancer Treatment: CRISPR can be employed to develop innovative cancer therapies (Cong et al., 2013; Hsu et al., 2015). One approach involves modifying a patient's own immune cells, such as T cells, using CRISPR to enhance their ability to recognize and kill cancer cells (Liu and Zhao,2018; Liu et al., 2023). This involves editing genes related to immune response and cell targeting. These modified cells can then be reintroduced into the patient to enhance the immune system's ability to fight cancer (Rivera and Jacks, 2015).

Infectious Disease Research: CRISPR has been utilized to study and combat various infectious diseases (Yuen et al., 2018; Bella et al., 2018). Researchers can use CRISPR to engineer animal models that mimic human responses to infections, aiding in the development of new treatments or vaccines (Cong et al.,2013; Liang et al., 2016). Additionally, CRISPR-based diagnostic tools are being developed to detect and identify specific pathogens quickly and accurately.

Antimicrobial resistance: CRISPR has the potential to address the rising problem of antimicrobial resistance. Researchers are investigating the use of CRISPR to target and disrupt specific genes responsible for antibiotic resistance in bacteria (Kim et al., 2016; Wilbie et al., 2019; Rodrigues et al., 2019). This could offer a new approach to combat drug-resistant bacteria and improve the effectiveness of existing antibiotics (Ghosh et al., 2019).

Viral Diseases: CRISPR has been explored as a potential strategy to combat viral infections, including those caused by HIV and herpes viruses (Kaminski et al.,2016; Yin et al., 2017; Dash et al., 2019). Scientists are investigating ways to use CRISPR to target and remove viral DNA from infected cells, potentially offering a cure for chronic viral diseases. 

Table 1: Applications of CRISPR technology in various disciplines.

The versatility of CRISPR technology allows it to be applied in numerous biotechnological areas. However, ethical considerations, regulatory frameworks, and responsible use of these technologies are essential to ensure their safe and beneficial application in Biotechnology. CRISPR technology has a wide range of applications in the field of biotechnology. Here are some key areas, where CRISPR is being utilized:

Drug discovery: CRISPR enables researchers to selectively disrupt or modify genes associated with specific diseases, providing valuable insights into the function of these genes. By studying the effects of gene perturbation, scientists can identify potential drug targets and develop more effective therapeutics. (Mou et al., 2015; Fagen et al., 2017; Nakamoto et al., 2018; Kawai et al., 2019).

Functional Genomics: CRISPR allows researchers to systematically study the function of individual genes and their interactions. By performing large-scale gene knockout or knockdown screens, scientists can identify genes that play crucial roles in biological processes, leading to a deeper understanding of complex biological systems (Zhou et al.,2014; Oliver and Gersbach, 2019).

Biomedical Research: CRISPR allows scientists to edit genes in human cells, offering new possibilities for studying disease mechanisms, developing disease models, and discovering potential therapies. By introducing specific mutations or correcting disease-causing mutations in human cell lines or organoids, researchers can gain insights into the molecular basis of diseases and explore novel treatments.

DNA and Genome Engineering: CRISPR technology can be used to manipulate DNA and genomes in a targeted manner (Hsu et al., 2013; Fu et al., 2013; Penget et al., 2014). It enables the precise addition, deletion, or modification of DNA sequences, making it valuable for genome engineering tasks such as creating transgenic organisms, modifying gene expression, or engineering cells for specific purposes (Ma et al., 2015).

Synthetic Biology: CRISPR can be used to engineer microorganisms for various purposes in synthetic biology (Wang et al., 2016; Jusiak et al., 2016). By modifying the genome of microorganisms, researchers can create custom-designed microbial factories capable of producing valuable compounds such as biofuels, pharmaceuticals, and industrial chemicals. CRISPR's precision and efficiency make it a powerful tool for genetic engineering in this context.

Environmental Applications: CRISPR can be applied to address environmental challenges. For example, it can be used to engineer microorganisms capable of degrading pollutants or cleaning up contaminated environments. Additionally, CRISPR-based technologies are being explored for monitoring and detecting environmental factors such as pollutants and pathogens (Stein et al., 2018).

Toxicity testing: Tests of toxicity are used to determine hazardous effects of a particular chemical which is absorbed in our body by various routes such as through oral, cutaneous, inhalation and circulation (Arya and Bist,2022). Toxicology is a constantly evolving field, especially in the area of developing alternatives to animal testing. Toxicological research must evolve and utilize adaptive technologies in an effort to improve public, environmental, an occupational health. The most commonly cited mechanisms of toxic action after exposure to a chemical can be revealed using CRISPR an emerging laboratory technique.

Agriculture

It is worth noting that the use of CRISPR in agriculture is subject to regulatory frameworks and public acceptance, which can vary across countries. The safety, potential off-target effects, and ethical considerations related to genetically modified organisms (GMOs) and gene editing technologies like CRISPR are topics of ongoing debate and scrutiny. The responsible and transparent application of CRISPR in agriculture is crucial to address these concerns and ensure the acceptance and benefit of these innovations. CRISPR technology has significant potential for applications in agriculture. Here are some key areas where CRISPR is being explored:

Crop Improvement: CRISPR can be used to introduce specific genetic changes in crops to improve their traits. This includes enhancing disease resistance, tolerance to environmental stresses such as drought or heat, increasing yield potential, and improving nutritional content (Abdelrahman et al.,2018; Tran et al.,2021). By targeting specific genes related to these traits, researchers can create desired modifications in crops more efficiently and precisely compared to traditional breeding methods.

Pest and Disease Control: CRISPR can help in developing crops that are more resistant to pests, diseases, and weeds. By targeting genes in pests or disease-causing organisms, scientists can potentially disrupt their biological processes, rendering them less harmful to crops (Zaidi et al., 2017; Aman et al., 2018; Zaidi et al.,2018; Chen et al., 2019). This approach offers an environmentally friendly alternative to conventional pesticide use and reduces the reliance on chemical interventions.

Sustainable Agriculture: CRISPR can contribute to more sustainable agricultural practices. By modifying crops to be more efficient in nutrient utilization, researchers aim to reduce fertilizer use and minimize nutrient runoff, which can have negative environmental impacts. CRISPR can also be used to engineer crops that require fewer resources, such as water, to thrive, thus promoting sustainable water management in agriculture (Chennakesavulu et al.,2022; Ceasar et al., 2022)

Quality and Shelf-Life Improvement: CRISPR can be utilized to enhance the quality and shelf-life of agricultural products. For example, researchers can modify genes involved in fruit ripening to slow down the process, allowing fruits to stay fresh for longer periods (Parmar, 2017; Sapkota and Knap,2020; Sharma et al., 2023). This can help reduce food waste and improve the quality of harvested crops.

Functional Foods: CRISPR can be employed to create crops with enhanced nutritional profiles, such as increased vitamin or mineral content (Ma et al., 2015; Lyzenga et al., 2019). This can help address nutrient deficiencies and improve the nutritional value of staple crops, particularly in regions where access to diverse diets is limited.

The translation of CRISPR-based therapies into clinical practice requires rigorous testing, safety assessments, and regulatory approvals. Ethical considerations and potential off-target effects of CRISPR editing also need to be carefully addressed to ensure the safety and efficacy of these medical applications. One of the primary concerns surrounding CRISPR is off-target effects, where unintended modifications occur in the genome. Innovative solutions such as high-fidelity Cas9 variants, base editing techniques, and novel delivery methods are being developed to enhance precision and reduce unintended genetic alterations. Another challenge lies in the ethical considerations surrounding germline editing and the potential for unintended consequences. The scientific community, policymakers, and society at large need to engage in thoughtful discussions to establish responsible guidelines and regulations for the use of CRISPR in human embryos or reproductive cells. Additionally, the accessibility and affordability of CRISPR technology are important factors to ensure its widespread adoption and benefit to all. Efforts are being made to simplify the technology, improve scalability, and reduce costs, allowing researchers from diverse backgrounds to leverage its potential. CRISPR's applications extend beyond human health, as it holds promise for addressing global challenges in agriculture, environmental conservation, and bioproduction. However, challenges such as delivery efficiency, tissue-specific targeting, and regulatory hurdles need to be addressed to fully utilize CRISPR's potential in these areas. Through continued research, innovation, and responsible use, we can unlock the full potential of CRISPR, shaping a future where genetic diseases can be cured, crops can be made more resilient, and our understanding of the genome can be deepened. The collaborative efforts of scientists, policymakers, and society will be instrumental in realizing the transformative power of CRISPR and ensuring its responsible and equitable application for the betterment of humanity.

The authors declare that they have no conflict of interest between them.

The authors are indebted to all mighty authority Godfather for giving us opportunity to write this manuscript for human welfare.

1. Aman R, Ali Z, Butt H, et al. (2018) RNA virus interference via CRISPR/Cas13a system in plants. Genome Biology 19:1.
2. Abdelrahman M, Abdullah MA, Aboughadareh AP, et al. (2018) Genome editing using CRISPR/Cas9–targeted mutagenesis: An opportunity for yield improvements of crop plants grown under environmental stresses. Plant Physiology and Biochemistry 131: 31-36.
3. Arya J, Bist R (2022) The diverse ways to determine experimental dose in animals. Hospice and Palliative Medicine International Journal 5: 21?24.
4. Bella R, Kaminski R, Mancuso P, et al. (2018) Removal of HIV DNA by CRISPR from patient blood engrafts in humanized mice. Molecular Therapy- Nucleic Acids 12: 275-282.
5. Bikard D, Euler CW, Jiang W, et al. (2014) Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nature Biotechnology 32: 1146-1150.
6. Ceasar SA, Maharajan T, Hillary VE (2022) Insights to improve the plant nutrient transport by CRISPR/Cas system. Biotechnology Advances 59: 107963.
7. Chen K, Wang Y, Zhang R, et al. (2019) CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture. Annual Review of Plant Biology 70: 667-697.
8. Chennakesavulu K, Singh H, Trivedi PK, et al. (2022) State-of-the-art in CRISPR technology and engineering drought, salinity, and thermo-tolerant crop plants. Plant Cell Report 41: 815–831.
9. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819-823.
10. Dash PK, Kaminski R, Bella R, et al. (2019) Sequential laser art and CRISPR treatments eliminate HIV-1 in a subset of infected humanized mice. Nature Communications 10: 2753.
11. Fagen JR, Collians D, Singh AK (2017) Sequential laser art and CRISPR treatments eliminate HIV-1 in a subset of infected humanized mice. Nature Communications 10: 2753.
12. Jusiak B, Cleto S, Perez-PP, et al. (2016) Engineering synthetic gene circuits in living cells with CRISPR technology. Trends in Biotechnology 34: 535-547.
13. Mojica FJM, Díez-Villaseñor C, Martiz JC, et al. (2005) Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. Journal of Molecular Evolution 60: 174-182.
14. Fu Y, Foden JA, Khayter C, et al. (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology 31: 822–826.
15. Gasiunas V, Barrangou R, Horvath P, et al. (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America 109: 2579-2586.
16. Ghosh C, Sarkar P, Issa R, et al. (2019) Alternatives to conventional antibiotics in the era of antimicrobial resistance. Trends Microbiology 27: 323-338.
17. Hsu PD, Scott DA, Weinstein JA, et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology 31: 827–832.
18. Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157: 1262-1278.
19. Kaminski R, Chen Y, Fischer T, et al. (2016) Elimination of HIV-1 genomes from human T-lymphoid cells by CRISPR/Cas9 gene editing. Scientific Report 6: 22555.
20. Kawai K, Negoro R, Ichikawa M, et al. (2019) Establishment of SLC15A1/PEPT1-knockout human-induced pluripotent stem cell line for intestinal drug absorption studies. Molecular Therapy- Methods Clinical Development 17: 49–57.
21. Kennedy EM, Kornepati AVR, Goldstein M, et al. (2014) Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease. Journal of Virology 88: 11965–11972.
22. Kim JS, Cho DH, Park M, et al. (2016) CRISPR/Cas9-mediated re-sensitization of antibiotic-resistant Escherichia coli harboring extended-spectrum beta-Lactamases. Journal of Microbiology Biotechnology 26: 394–401.
23. Lander ES (2016) The Heroes of CRISPR. Cell 164: 18–28.
24. Liang X, Sun L, YuT, et al. (2016) A CRISPR/Cas9 and Cre/Lox system-based express vaccine development strategy against re-emerging Pseudorabies virus. Sci Rep 6: 19176.
25. Liu X, Zhao Y. (2018) CRISPR/Cas9 genome editing: fueling the revolution in cancer immunotherapy. Curr Res Transl Med 66: 39–42.
26. Liu Z, Shi M, Ren Y, et al. (2023) Recent advances and applications of CRISPR-Cas9 in cancer immunotherapy. Molecular Cancer 22-35.
27. Li Q, Sapkota M, Knaap EVD (2020) Perspectives of CRISPR/Cas-mediated cis-engineering in horticulture: Unlocking the neglected potential for crop improvement. Horticulture Research 7: 36.
28. Lyzenga WJ, Harrington M, Bekkaoui D, et al. (2019) CRISPR/Cas9 editing of three CRUCIFERIN C homoeologues alters the seed protein profile in Camelina sativa. BMC Plant Biology 19: 292.
29. Ma X, Zhang Q, Zhu Q, et al. (2015) A Robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant 8: 1274-1284.
30. Jinek M, Chylinski K, Fonfara I, et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816-821.
31. Mishra A, Bist R, Sharma V (2019) Study of pervasiveness, antimicrobial vulnerability and resolution of best method for extended spectrum beta lactamases Escherichia coli isolates. Hospice and Palliative Medicine International Journal 3: 59-64.
32. Nakamoto FK, Okamoto S, Mitsui J, et al. (2018) The pathogenesis linked to coenzyme Q10 insufficiency in iPSC-derived neurons from patients with multiple-system atrophy. Scientific Report 8: 14215.
33. Nguyen TM, Zhang Y, Pandolfi PP. Virus against viruses: a potential treatment for 2019-nCov (SARS-CoV-2) and other RNA viruses. Cell Research 30: 189-190.
34. Parmar N, Singh KH, Sharma D, et al. (2017) Genetic engineering strategies for biotic and abiotic stress tolerance and quality enhancement in horticultural crops: A comprehensive review. Biotechnology 7: 239.
35. Peng Y, Clark KJ, Campbell JM, Panetta MR, et al. (2014) Making designer mutants in model organisms. Development 141: 4042–4054.
36. Oliver PA, Gersbach CA (2019) The next generation of CRISPR–Cas technologies and applications. Nature Reviews Molecular Cell Biolology 20: 490–507.
37. Rivera SFJ, Jacks T (2015) Applications of the CRIWSPR-Cas 9 system in cancer biology. Nature Reviews Cancer 15: 387-393.
38. Rodrigues M, McBride SW, Hullahalli K, et al. (2019) Conjugative Delivery of CRISPR-Cas9 for the Selective Depletion of Antibiotic-Resistant Enterococci. Antimicrobial Agents Chemotherapy 63: 1454-1419.  
39. Sharma P, Pandey A, Malviya R, et al. (2023) Genome editing for improving nutritional quality, post-harvest shelf life and stress tolerance of fruits, vegetables, and ornamentals. Frontiers in Genome Edditing 24: 1094965.
40. Song B, Fan Y, He W, et al. (2015) Improved hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system. Stem Cells and Development 24: 1053–1065.
41. Stein HP, Navajas-Pérez R, Aranda E. Potential for CRISPR Genetic Engineering to Increase Xenobiotic Degradation Capacities in Model Fungi. In: Prasad, R., Aranda, E. (eds) Approaches in Bioremediation. Nanotechnology in the Life Sciences. Springer, Cham, 2018. Potential for CRISPR Genetic Engineering to Increase Xenobiotic Degradation Capacities in Model FungiIn: Ram Prasad, Elisabet Aranda (Eds.), Approaches in bioremediation Nanotechnology in the Life Sciences 2018: 61-78.
42. Tran MT, Doan DTH, Kim J, et al. (2021) CRISPR/Cas9-based precise excision of SlHyPRP1 domain(s) to obtain salt stress-tolerant tomato. Plant Cell Reports 40: 999-1011.
43. Yuen K-S, Wang Z-M, Wong N-HM, et al. (2018) Suppression of epstein-barr virus DNA load in latently infected nasopharyngeal carcinoma cells by CRISPR/Cas9. Virus Research 244: 296–303.
44. Wilbie D, Walther J, Mastrobattista E (2019) Delivery Aspects of CRISPR/Cas for in Vivo Genome Editing. Accounts of Chemical Research 52: 1555-1564.
45. Xie F, Ye L, Chang JC, et al. (2014) Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Research 24:1526–1533.
46. Yin C, Zhang T, Qu X, et al. (2017) In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models. Molecular Therapy 25: 1168-1186.
47. Yi Wang, Zhang ZT, Seo SO, et al. (2016) Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification, and Desirable “Clean” Mutant Selection in Clostridium beijerinckii as an Example. ACS Synthetic Biology 5: 721-732.
48. Zaidi SS, Mahfouz MM, Mansoor S (2017) CRISPR-Cpf1: A New Tool for Plant Genome Editing. Trends Plant Sci 22: 550-553.
49. Zaidi SS, Mukhtar MS, Mansoor S (2018) Genome editing: Targeting susceptibility genes for plant disease resistance. Trends Biotechnol 36: 898-906.
50. Zhou Y, Zhu S, Cai C, et al. (2014) High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509: 487-491.