CRISPR/Cas 9 Gene Editing

Abstract

This review paper aims to examine gene editing, specifically the CRISPR/Cas9 technology. The mechanisms, capabilities, and applications of this gene editing tool are explored. CRISPR/Cas9 is known for being one of the leading molecular techniques utilized to analyze and manipulate DNA; however, it has its complications in terms of ethical practices and modifications to its technology. 

 

Introduction

Genome editing is a modifying tool that allows scientists to change an organism’s DNA by targeting a specific target site of the genome. Deletion, replacement, and insertion of the DNA can occur with gene editing (Makarova & Koonin, 2015)(Ebrahimi & Hashemi, 2020). Several methods of gene editing have been developed. Three genome editing tools currently exist: ZNFs, TALENs, and CRISPR/Cas systems (Nemudryi et.al., 2014). These technologies use the approach of intentionally breaking a specific location in the DNA. Known as a double-strand break (DSB), a cut is made, and when cells repair said cut scientists can modify and make exact changes to the region (Makarova et.al., 2019)(Krishan et.al., 2016). Despite its potential, the CRISPR/Cas9 system faces challenges, but it remains the most accurate and cost-effective genetic engineering tool compared to ZFNs and TALENs, making it widely applied in various research areas, including cancer studies and gene manipulation. Its simpler assembly and higher efficiency allow for precise genetic modifications and treatments for genetic disorders.

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Following the development of TALENs, another type of genome editing tool that was discovered was the CRISPR/Cas system (clustered regulatory interspaced short palindromic repeats). Bacteria and archaea themselves possess this system as a defense mechanism against viruses (Ran et.al., 2013). The system’s immune response occurs in three steps: adaptive, expression, and interference phases. This system relies on small RNAs to identify and silence the foreign DNA. When a virus attacks, the organism adds a piece of the virus’s DNA to its DNA. This system has cas genes organized in operons and a CRISPR array that contains unique sequences mixed with repetitive ones. The organism can later recognize the virus because of this. RNA molecules are made in this spot to help recognize and bind to the DNA of the virus. Then enzymes come in to cut the DNA of the virus to prevent it from causing harm (Ran et.al., 2013). In the adaptive phase, bacteria and archaea incorporate short fragments of foreign DNA known as protospacers into their DNA. This occurs at the beginning of the CRISPR array. In the expression and inference phrase, the CRISPR array is transcribed into precursor RNA molecules, which cleave to make short CRISPR RNAs (crRNAs). The crRNAs bind to complementary photospacer sequences of plasmid targets or viral DNA. crRNAs recognize these sequences and silence the foreign DNA by utilizing Cas proteins (Ran et.al., 2013). Unlike the TALEN proteins, the CRISPR/Cas system identifies its target site DNA through a matching method between non-coding DNA and RNA. A combination of non-coding RNA and Cas proteins forms a complex (Chen et.al., 2020). The CRISPR/Cas system has two class types of systems. The classification largely depends on the composition of the protein and genetic variation of the complexes (Ran et.al., 2013).

 

How CRISPR / Cas9 Works

The CRISPR/Cas9 system specifically is a subtype that utilizes a Cas9 protein deriving from Streptococcus pyogenes (S. pyogenes) (Rasul et.al., 2022). It’s part of the type II CRISPR/Cas systems, known as “HNH” systems (Stroik, n.d.). Type II CRISPR/Cas loci, along with the cas9 gene, contain ubiquitous cas1 and cas2 genes. Most of these loci also have one or two genes for tracrRNA, an RNA partly homologous to the cognate CRISPR. These systems rely on RNase III and tracrRNA to process the pre-crRNA. The Cas9 protein contains two nuclease domains: the RuvC-like nuclease domain and the HNH (McrA-like) nuclease domain, located in the center of the protein. Both nuclease domains are needed for the Cas9 enzyme to cleave the target DNA effectively (Stroik, n.d.). The type II CRISPR/Cas system is composed of the Cas9 protein guided by RNA and a single-guide RNA (sgRNA). The two cutting parts that the cas9 protein contains, HNH and RuvC, chop one side of the target DNA. The sgRNA is a ‘simpler’ version of two types of RNA, tracrRNA and crRNA. When the Cas9 protein and sgRNA come together, they form a Cas9 ribonucleoprotein (RNP). The ribonucleoprotein can bind and cleave to the DNA target of interest. In addition, a specific DNA sequence called a protospacer adjacent motif (PAM) is needed by the Cas9 protein to bind to the target DNA (Ebrahimi & Hashemi, 2020). The presence of a specific protospacer adjacent motif (PAM) is needed for Cas9 target site recognition (Nemudryi et.al., 2014).  Similarly to the other two gene editing tools, Cas9 conducts editing by causing a double-strand break (DSB) at a particular genome site. Two methods can be used to repair: one called the homology-directed repair (HDR) or the error-prone non-homologous end joining (NHEJ) pathways. NHEJ is more efficient than HDR but might introduce mistakes, like random insertions or deletions of DNA pieces, which can turn off the targeted genes due to the makeshift of mutations. With HDR, it can introduce accurate DNA changes by utilizing a matching DNA template (figure 3). Changes to the genome can be made with the use of sgRNAs, like the deletion and knockout of genes (Ran et.al., 2013)(Gaj et.al., 2016).

 

Barriers to Widespread Use & Modifications

Applying CRISPR/Cas9 to in vivo use, scientists have noted an “occurrence of off-targeting modifications, the possibility of causing autoimmune disorders, the identification of a proper delivery technique, and, lastly, ethical concerns” are issues they’ve been faced with (Das et.al., 2019). Cas9 has been seen to cause negative effects, like disruptions to the genes of the cells being studied (Ng, 2020). Scientists need to carefully monitor to minimize these effects. Although it’s been seen that the CRISPR/Cas9 system is much more effective with in vitro research than with in vivo, it still faces similar problems, just not at a higher risk. 

 

Scientists have developed several solutions for when off-target activity occurs when using the CRISPR/Cas9 system: (1) Modifying the guide RNA (gRNA). This is done by adding guanine nucleotides to the 5’ end or shortening the 3’. (2) Using a pair of “nickase “ Cas9 enzymes instead of the regular nuclease can produce more accurate cuts and less off-target effects. (3) Using lower Cas9-gRNA complex concentrations can reduce unnecessary off-target cleavage. (4) Utilizing tru-gRNAs that are truncated at the 5’ and are more sensitive to mismatches can decrease off-target mutagenesis. In addition, software has been developed so scientists can design gRNA with a low probability of off-target effects. They also analyze the genome and generate sgRNAs to get the desired target. These methods can help minimize mistakes that the CRISPR/Cas9 system may induce (Urnov et.al., 2010).

 

Ethical Concerns, Existing & Future Applications and Conclusion

CRISPR/Cas9's ability to turn off and on genes has allowed scientists to study genetic diseases such as cancer to see why cells become cancerous. Plants and animals can be altered to get desirable traits, like bigger fruit or more muscle. It’s a bit more complicated with humans and altering their genomes to delete mutations that cause disease or enhance the characteristics of their offspring. Genetic modifications are not passed down to offspring if it’s made to non-reproductive cells, like somatic cells. But if alterations are made to germ cells - that turn into eggs, sperm, or embryos - those changes can be inherited by the offspring (Redman et.al., 2016). This ability to transform genes is significant and opens up the possibility of altering genetic characteristics that can be passed on to generations. 

It also raises ethical concerns about inheritable genetic modifications. In 2015, Chinese scientists utilized CRISPR/Cas9 to edit the HBB (human beta-globin) gene in human zygotes. Questions were raised about whether CRISPR would enable the creation of “designer babies,” where people select desirable genetic traits. Concerns about who would have the authority to decide the genetic makeup of children born using this technology and whether a child has the autonomy and consent for this. There are also worries that people are going to use this to enhance human abilities beyond just treating disease. Once it’s out of the laboratory and into the market, it would make regulating this genetic tool much more difficult. 

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Off-target mutations are a major concern with CRISPR/Cas9. Although the technology has been highly praised for its simple structure and ease of genetic editing (compared to other tools like ZFNs and TALENs, which require specific proteins to bind to DNA and can take years to design, CRISPR/Cas9 just needs a complementary RNA strand), it can lead to “deleterious effects on humans and the environment”(Redman et.al., 2016). Researchers pointed out that this effect is higher in humans compared to other organisms. Cell death and abnormal transformations can occur due to mutations. Modifications to cells that are harder to infect need to be precise for the editing to work and be accepted. The integrity of the environment can be disrupted due to off-target mutations since they can be transferred to other organisms. This is known as gene drive.

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Conclusion

CRISPR/Cas9 technology has the potential to treat diseases via genetic manipulation. As mentioned, one of the many illnesses studied using CRISPR/Cas9 is cancer (Jinek et.al., 2012). Cancerous cells can be silenced and tumor suppressor genes can be overexpressed. Liver, cardiovascular, and other diseases can potentially be treated with this technology. The illnesses currently being studied with the application of CRISPR/Cas9 are HIV, Sickle Cell, coronaviruses, and others (Jinek et.al., 2012). In all, this suggests that the construction of an effective genome editing tool that can be delivered to the human body can be accomplished. Future research is improving off-target mutations, conducting precise manipulation, and designing better CRISPR/Cas systems. Off-target mutations and other mistakes can lead to major consequences that can affect humans and other organisms. Carefully identifying and improving on these mistakes is vital before the technology is utilized in clinical settings. The CRISPR/Cas9 technology will continue to expand the knowledge in molecular biology, hoping to improve the understanding of genetic diseases to develop effective therapies. CRISPR/Cas9 holds the ability to revolutionize medicine for a wide range of illnesses. With ongoing research and awareness of ethics and safety, this technology can transform the future of healthcare and more. Nonetheless, scientists will likely explore CRISPR-Cas9 applications in germline cells, and once regulatory frameworks are established, they will be integrated into medical practices and other research. It’s vital to implement legal, ethical, and social regulations while the research on CRISPR/Cas9 for clinical purposes continues (Ayanoglu et.al., 2020).

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References

Ayanoglu, F. B., Elcin, A. E., & Elcin, Y. M. (2020). Bioethical Issues in Genome Editing by the CRISPR/Cas9 Technology. Turkish Journal of Biology, 44(2). https://doi.org/10.3906/biy-1912-52 

​

Chen, Y., Jiang, H., Wang, T., He, D., Tian, R., Cui, Z., Tian, X., Gao, Q., Ma, X., Yang, J., Wu, J., Tan, S., Xu, H., Tang, X., Wang, Y., Yu, Z., Han, H., Das, B. C., Severinov, K., & Hitzeroth, I. I. (2020). In vitro and in vivo growth inhibition of human cervical cancer cells via human papillomavirus E6/E7 mRNAs’ cleavage by CRISPR/Cas13a system. Antiviral Research, 178, 104794. https://doi.org/10.1016/j.antiviral.2020.104794 

 

Das, J., Bhatia, P., & Singh, A. (2019). CRISP Points on Establishing CRISPR-Cas9 In Vitro Culture Experiments in a Resource Constraint Haematology Oncology Research Lab. Indian Journal of Hematology & Blood Transfusion : An Of icial Journal of Indian Society of Hematology and Blood Transfusion, 35(2), 208–214. https://doi.org/10.1007/s12288-018-1008-z 

 

Ebrahimi, V., & Hashemi, A. (2020). Challenges of In vitro Genome Editing with CRISPR/Cas9 and Possible Solutions: A Review. Gene, 144813. https://doi.org/10.1016/j.gene.2020.144813 

 

Gaj, T., Sirk, S. J., Shui, S., & Liu, J. (2016). Genome-Editing Technologies: Principles and Applications. Cold Spring Harbor Perspectives in Biology, 8(12), a023754. https://doi.org/10.1101/cshperspect.a023754 

 

Gostimskaya, I. (2022). CRISPR–Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing. Biochemistry (Moscow), 87(8), 777–788. https://doi.org/10.1134/s0006297922080090 

 

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(6096), 816–821. https://doi.org/10.1126/science.1225829 

 

Joung, J. K., & Sander, J. D. (2012). TALENs: a widely applicable technology for targeted genome editing. Nature Reviews Molecular Cell Biology, 14(1), 49–55. https://doi.org/10.1038/nrm3486 

 

Krishan, K., Kanchan, T., & Singh, B. (2016). Human Genome Editing and Ethical Considerations. Science and Engineering Ethics, 22(2), 597–599. https://doi.org/10.1007/s11948-015-9675-8 

 

Li, T., Yang, Y., Qi, H., Cui, W., Zhang, L., Fu, X., He, X., Liu, M., Li, P., & Yu, T. (2023). CRISPR/Cas9 therapeutics: progress and prospects. Signal Transduction and Targeted Therapy, 8(1), 1–23. https://doi.org/10.1038/s41392-023-01309-7

 

Makarova, K. S., & Koonin, E. V. (2015). Annotation and Classification of CRISPR-Cas Systems. Methods in Molecular Biology, 1311, 47–75. https://doi.org/10.1007/978-1-4939-2687-9_4 

 

Makarova, K. S., Wolf, Y. I., Iranzo, J., Shmakov, S. A., Alkhnbashi, O. S., Brouns, S. J. J., Charpentier, E., Cheng, D., Haft, D. H., Horvath, P., Moineau, S., Mojica, F. J. M., Scott, D., Shah, S.A., Siksnys, V., Terns, M. P., Venclovas, Č., White, M. F., Yakunin, A. F., & Yan, W.(2019). Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org/10.1038/s41579-019-0299-x

 

MedlinePlus. (2022, March 22). What Are Genome Editing and CRISPR-Cas9? Medlineplus.gov; Medlineplus. https://medlineplus.gov/genetics/understanding/genomicresearch/genomeediting/

 

Mehravar, M., Shirazi, A., Mehrazar, M. M., & Nazari, M. (2019). In Vitro Pre-validation of Gene Editing by CRISPR/Cas9 Ribonucleoprotein. Avicenna Journal of Medical Biotechnology, 11(3), 259–263.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626505/#:~:text=Therefore%2C%20in %20vitro%20digestion%20of 

 

Nemudryi, A. A., Valetdinova, K. R., Medvedev, S. P., & Zakian, S. M. (2014). TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery. Acta Naturae, 6(3), 19–40. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207558/ 

 

Ng, D. (2020). A Brief History of CRISPR-Cas9 Genome-Editing Tools. Bitesize Bio. https://bitesizebio.com/47927/history-crispr/

 

Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 8(11), 2281–2308. https://doi.org/10.1038/nprot.2013.143 

 

Rasul, M. F., Hussen, B. M., Salihi, A., Ismael, B. S., Jalal, P. J., Zanichelli, A., Jamali, E., Baniahmad, A., Ghafouri-Fard, S., Basiri, A., & Taheri, M. (2022). Strategies to overcome the main challenges of the use of CRISPR/Cas9 as a replacement for cancer therapy. Molecular Cancer, 21(1). https://doi.org/10.1186/s12943-021-01487-4 

 

Redman, M., King, A., Watson, C., & King, D. (2016). What Is CRISPR/Cas9? Archives of Disease in Childhood - Education & Practice Edition, 101(4), 213–215. https://doi.org/10.1136/archdischild-2016-310459 

 

Shinwari, Z. K., Tanveer, F., & Khalil, A. T. (2018). Ethical Issues Regarding CRISPR Mediated Genome Editing. Current Issues in Molecular Biology, 26(1), 103–110. https://doi.org/10.21775/cimb.026.103 

 

Stroik, S. (n.d.). CRISPR 101: Cas9 vs. The Other Cas(s). Blog.addgene.org. https://blog.addgene.org/crispr-101-cas9-vs.-the-other-cass 

 

TALEN Technology Information - US. (n.d.). Www.thermofisher.com. https://www.thermofisher.com/us/en/home/life-science/genome-editing/genome-editing-learning center/designer-talen-technology-information.html 

 

Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S., & Gregory, P. D. (2010). Genome editing with engineered zinc finger nucleases. Nature Reviews Genetics, 11(9), 636–646. https://doi.org/10.1038/nrg2842 

 

Xu, Y., & Li, Z. (2020). CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy. Computational and Structural Biotechnology Journal, 18(18), 2401–2415. https://doi.org/10.1016/j.csbj.2020.08.031