CRISPR/Cas9 as a tool for Genome Editing: A Mini Review on Development and Approaches

  • Elham Riazimontazer Biotechnology Research Center
  • Ahmad Gholami Biotechnology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
Keywords: CRISPR/Cas9, Genome editing, Streptococcus pyogenes, CrRNA, Nuclease


By introducing recombinant DNA technology, first reported in 1972, biological researchers were able to manipulate DNA molecules to develop new therapeutic strategies. Nowadays, targeted methods for engineering the genome of diverse organisms have provided a powerful tool in the treatment of genetic diseases. Clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein 9 (CRISPR/Cas9) technique is one of the newest genome modification tools based on prokaryotic adaptive immune system. The simplicity and flexibility of the CRISPR/Cas9 site-specific nuclease system and its efficiency has led to its widespread use in many biological research areas. In this review, the basis of this technique and its application in the treatment of genetic diseases are explored while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, CRISPR/Cas9 is used widely as innovative scaffold from basic biology to biotechnology and medicine.


1. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262-78.
2. Lino CA, Harper JC, Carney JP, Timlin JA. Delivering CRISPR: a review of the challenges and approaches. Drug delivery. 2018;25(1):1234-57.
3. Jiang F, Doudna JA. CRISPR–Cas9 structures and mechanisms. Annual review of biophysics. 2017;46:505-29.
4. Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology. 2005;151(8):2551-61.
5. Mojica FJ, Díez-Villaseñor C, García-Martínez J, Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology. 2009;155(3):733-40.
6. Deveau H, Barrangou R, Garneau JE, Labonté J, Fremaux C, Boyaval P, et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of bacteriology. 2008;190(4):1390-400.
7. Horvath P, Romero DA, Coûté-Monvoisin A-C, Richards M, Deveau H, Moineau S, et al. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. Journal of bacteriology. 2008;190(4):1401-12.
8. Mir A, Edraki A, Lee J, Sontheimer EJ. Type II-C CRISPR-Cas9 biology, mechanism, and application. ACS chemical biology. 2018;13(2):357-65.
9. Amitai G, Sorek R. CRISPR–Cas adaptation: insights into the mechanism of action. Nature Reviews Microbiology. 2016;14(2):67.
10. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007;315(5819):1709-12.
11. Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biology direct. 2006;1(1):7.
12. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011;471(7340):602-7.
13. Thurtle‐Schmidt DM, Lo TW. Molecular biology at the cutting edge: a review on CRISPR/CAS9 gene editing for undergraduates. Biochemistry and Molecular Biology Education. 2018;46(2):195-205.
14. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences. 2012;109(39):E2579-E86.
15. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. science. 2012;337(6096):816-21.
16. Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. Journal of Biological Chemistry. 2014;289(19):13284-94.
17. Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature biotechnology. 2013;31(9):833-8.
18. Ran FA, Hsu PD, Lin C-Y, Gootenberg JS, Konermann S, Trevino AE, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154(6):1380-9.
19. Lieber MR. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annual review of biochemistry. 2010;79:181-211.
20. San Filippo J, Sung P, Klein H. Mechanism of eukaryotic homologous recombination. Annu Rev Biochem. 2008;77:229-57.
21. Zaboikin M, Zaboikina T, Freter C, Srinivasakumar N. Non-homologous end joining and homology directed DNA repair frequency of double-stranded breaks introduced by genome editing reagents. PloS one. 2017;12(1):e0169931.
22. Doudna JA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346(6213).
23. Xiao-Jie L, Hui-Ying X, Zun-Ping K, Jin-Lian C, Li-Juan J. CRISPR-Cas9: a new and promising player in gene therapy. Journal of medical genetics. 2015;52(5):289-96.
24. Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nature methods. 2013;10(11):1116-21.
25. Kleinstiver BP, Prew MS, Tsai SQ, Nguyen NT, Topkar VV, Zheng Z, et al. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nature biotechnology. 2015;33(12):1293-8.
26. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015;523(7561):481-5.
27. DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research. 2013;41(7):4336-43.
28. Li J-F, Norville JE, Aach J, McCormack M, Zhang D, Bush J, et al. Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature biotechnology. 2013;31(8):688-91.
29. Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nature biotechnology. 2013;31(8):691-3.
30. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819-23.
31. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910-8.
32. Dow LE. Modeling disease in vivo with CRISPR/Cas9. Trends in molecular medicine. 2015;21(10):609-21.
33. Choi PS, Meyerson M. Targeted genomic rearrangements using CRISPR/Cas technology. Nature communications. 2014;5(1):1-6.
34. Maddalo D, Manchado E, Concepcion CP, Bonetti C, Vidigal JA, Han Y-C, et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature. 2014;516(7531):423-7.
35. Barrangou R, May AP. Unraveling the potential of CRISPR-Cas9 for gene therapy. Taylor & Francis; 2015.
36. Ebina H, Misawa N, Kanemura Y, Koyanagi Y. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Scientific reports. 2013;3:2510.
37. Hu W, Kaminski R, Yang F, Zhang Y, Cosentino L, Li F, et al. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. Proceedings of the National Academy of Sciences. 2014;111(31):11461-6.
38. Liu X, Hao R, Chen S, Guo D, Chen Y. Inhibition of hepatitis B virus by the CRISPR/Cas9 system via targeting the conserved regions of the viral genome. Journal of General Virology. 2015;96(8):2252-61.
39. Seeger C, Sohn JA. Targeting hepatitis B virus with CRISPR/Cas9. Molecular Therapy-Nucleic Acids. 2014;3:e216.
40. Zhen S, Hua L, Takahashi Y, Narita S, Liu Y-H, Li Y. In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by CRISPR/Cas9. Biochemical and biophysical research communications. 2014;450(4):1422-6.
41. Wang J, Quake SR. RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection. Proceedings of the National Academy of Sciences. 2014;111(36):13157-62.
42. Yin H, Xue W, Chen S, Bogorad RL, Benedetti E, Grompe M, et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nature biotechnology. 2014;32(6):551-3.
43. Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN. Prevention of muscular dystrophy in mice by CRISPR/Cas9–mediated editing of germline DNA. Science. 2014;345(6201):1184-8.
44. Schwank G, Koo B-K, Sasselli V, Dekkers JF, Heo I, Demircan T, et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell stem cell. 2013;13(6):653-8.
45. Li HL, Fujimoto N, Sasakawa N, Shirai S, Ohkame T, Sakuma T, et al. Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9. Stem cell reports. 2015;4(1):143-54.
46. Wu Y, Zhou H, Fan X, Zhang Y, Zhang M, Wang Y, et al. Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells. Cell research. 2015;25(1):67-79.
47. Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell stem cell. 2013;13(6):659-62.
48. Xie F, Ye L, Chang JC, Beyer AI, Wang J, Muench MO, et al. Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome research. 2014;24(9):1526-33.
49. Smith C, Abalde-Atristain L, He C, Brodsky BR, Braunstein EM, Chaudhari P, et al. Efficient and allele-specific genome editing of disease loci in human iPSCs. Molecular Therapy. 2015;23(3):570-7.
50. Musunuru K. The hope and hype of CRISPR-Cas9 genome editing: a review. JAMA cardiology. 2017;2(8):914-9.
51. Jaganathan D, Ramasamy K, Sellamuthu G, Jayabalan S, Venkataraman G. CRISPR for crop improvement: an update review. Frontiers in plant science. 2018;9:985.
52. Wang H, La Russa M, Qi LS. CRISPR/Cas9 in genome editing and beyond. Annual review of biochemistry. 2016;85:227-64.
53. Cui Y, Xu J, Cheng M, Liao X, Peng S. Review of CRISPR/Cas9 sgRNA design tools. Interdisciplinary Sciences: Computational Life Sciences. 2018;10(2):455-65.
54. Dara M, Talebzadeh M. CRISPR/Cas as a Potential Diagnosis Technique for COVID-19. Avicenna journal of medical biotechnology. 2020;12(3):201-2.
55. Nalawansha DA, Samarasinghe KT. Double-Barreled CRISPR Technology as a Novel Treatment Strategy For COVID-19. ACS Pharmacology & Translational Science. 2020;3(5):790-800.