Using CRISPR-Cas9 for Therapeutic Protein Production (Review Article)


  • Ashraf Ullah Khan Faculty of Pharmacy and Health Sciences, University of Balochistan, Quetta, Pakistan
  • Zabi Ullah Faculty of Curative Medicine, Malalay Institute of Higher Education, Aino Maina, Kandahar, Afghanistan



CRISPR/Cas9, genome editing, gene therapy, sgRNA


Existence of CRISPR/Cas9 systems in bacteria and archaea has been noted to be the reason for these organisms’ ability to disarm invading nucleic acids. Such immunity is noted to arise from the targeting of the invading nucleic acids by guiding RNAs (sgRNAs), their cleavage by Cas9 (an endonuclease), and their subsequent integration into CRISPR locus. Recent studies have shown that the CRISPR/Cas9 tool can be adopted for gene editing in eukaryotic cells and thus offering potential for its use to treat genetic conditions. In this review, CRISPR/Cas9 has been shown to be an effective genome-editing tool with studies showing efficacy in zygote editing, in-vivo editing of somatic cells and ex-vivo editing of somatic cells. Occurrence of off-target effects however make zygote editing in human cells ethically questionable due to possibility of introducing unwanted mutations that may be passed on to the progeny. Nevertheless, observations that such off-target effects arise mainly from the promiscuity of sgRNAs rather that errors in CRISPR/Cas9 system show promise for increased specificity by developing better sgRNAs.  Such increased specificity will facilitate the adoption of CRISPR/Cas9 for clinical use in treatment of conditions such as β-thalassemia, cystic fibrosis, Duchenne muscular dystrophy and HIV.

Author Biographies

Ashraf Ullah Khan, Faculty of Pharmacy and Health Sciences, University of Balochistan, Quetta, Pakistan

Faculty of Pharmacy and Health Sciences, University of Balochistan, Sariab Road, Quetta (Pakistan), P.O Box 87300, Quetta, Pakistan.

Zabi Ullah, Faculty of Curative Medicine, Malalay Institute of Higher Education, Aino Maina, Kandahar, Afghanistan

Faculty of Curative Medicine, Malalay Institute of Higher Education, Kandahar, Afghanistan.


Genome editing for all. (2014). Nature Biotechnology, 32(4), 295. doi:10.1038/nbt.2879

Lee, J. S., Grav, L. M., Lewis, N. E., & Kildegaard, H. F. (2015). CRISPR/ Cas9-mediated genome engineering of CHO cell factories: Applications and perspectives. Biotechnology Journal, 10(7), 979-94. doi:10.1002/biot.201500080032

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337, 816-821. doi:10.1126/science.1225829

Savić, N., & Schwank, G. (2016). Advances in the therapeutic CRISPR/Cas9 genome editing. Translational Research, 168(), 15-21.

Lanphier, E., & Urnov, F. (2015). Don’t edit the human germ line. Nature, 519, 410-411. Retrieved from!/menu/main/topColumns/topLeftColumn/pdf/519410a.pdf

Reardon, S. (2015). Gene-editing wave hits clinic. Nature, 527, 146-147. Retrieved from!/menu/main/topColumns/topLeftColumn/pdf/nature.2015.18737.pdf

Callaway, E. (2016). Embryo editing gets green light. Nature, 530, 18. Retrieved from!/menu/main/topColumns/topLeftColumn/pdf/nature.2016.19270.pdf

Wang, H., Yang, H., Shivalila, C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F., & Jaenisch, R. (2013). One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell, 153(4), 910-918. doi:10.1016/j.cell.2013.04.025.

Wu, Y., Liang, D., Wang, Y., Bai, M., Tang, W., Bao, S., … Li, J. (2013). Correction of a genetic disease in muse via use of CRISPR-Cas9. Cell Stem Cell, 13(6), 659-662.

Cyranoski, D. (2015). Embryo editing divides scientists. Nature, 519, 272. Retrieved from!/menu/main/topColumns/topLeftColumn/pdf/519272a.pdf

Liang, P., Xu, Y., Zhang, X., Ding, C.,Huang, R., Zhang, Z., … Huang, J. (2015). CRISPR/Cas9-mediated gene editing in human triponuclear zygotes. Protein Cell, 6(5), 363-372. doi:10.1007/s13238-015-0153-5

Yin, H., Xue, W., Chen, S., Bogorad, R., Benedetti, E., Grompe, M., Koteliansky, V., … Anderson, D. G. (2014). Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nature Biotechnology, 32(6), 551-3. doi:10.1038/nbt.2884

Nelson, D. L., & Cox, M. M. (2004). Lehninger principles of biochemistry (4th ed.). New York: W. H. Freeman & Company

Ding, Q., Strong, A., Patel, K. M., Ng, S-L., Gosis, B. S., Regan, S. N., … Musunuru, K. (2014). Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circulation Research, 115(5), 488-492, doi:10.1161/CIRCRESAHA.115.304351

Lin, S. R., Yang, H. C., Kuo, Y. T., Liu, C. J., Yang, T. Y., Sung, K. C., … Chen, P. J. (2014). Molecular Therapy. Nucleic Acids, 19(3), e186. doi:10.1038/mtna.2014.38.

Xie, F., Ye, L., Chang, J. C., Beyer, A. I., Wang, J., … Kan, Y. W. (2014). Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Research, 24(9), 1526-33, doi:10.1101/gr.173427.114.

Schwank, G., Koo, B. K., Sasselli, V., Dekkers, J. F., Heo, I., Demircan, T., Clevers, H. (2013). Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell, 13(6), 653-8. doi:10.1016/j.stem.2013.11.002.

Li, H. L., Fujimoto, N., Sasakawa, N., Shirai, S., Ohkame, T., Sakuma, T., … Hotta, A. (2015). Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9. Stem Cell Reports, 4(1):143-54. doi: 10.1016/j.stemcr.2014.10.013.

Ousterout, D. G., Kabadi, A. M., Thakore, P. I., Majoros, W. H., Reddy, T. E., & Gersbach, C. A. (2015). Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nature Communications, 6, 6244. doi: 10.1038/ncomms7244

O’Green, H., Yu, A. S., & Segal, D. J. (2015). How specific is CRISPR/Cas9 really? Current Opinion in Chemical Biology, 29, 72-78.




How to Cite

Khan, A. U., & Ullah, Z. (2021). Using CRISPR-Cas9 for Therapeutic Protein Production (Review Article). Asian Journal of Pharmacy, Nursing and Medical Sciences, 9(1).