An Introduction To CRISPR

Introduction

The discovery of CRISPR is a significant scientific breakthrough in the field of genomics. CRISPR which is abbreviated as “Clustered Regularly Interspaced Short Palindromic Repeats”, was first observed in the genome of E.coli bacteria, and has shown strange repetitive sequences of DNA. After decades of research, it eventually became evident that the CRISPR-Cas9 complex was utilized in Prokaryotes and was used as a form of defense mechanism against Viral Infections. This finding of the Prokaryotic defense mechanism eventually led to the development & advancements of genome editing in Eukaryotes using CRISPR-Cas9. From in-vivo treatments for genetic conditions to the induction of fetal hemoglobin in sickle-cell patients, CRISPR-Cas9 demonstrated promising results in clinical trials. However, ethical considerations raise concerns regarding unintended consequences and equitable access to the treatment. This article delves into the history, mechanism, current research, and ethical considerations of CRISPR-Cas9.

Discovery of CRISPR

In 1987, a group of researchers at Osaka University observed clusters of unusual repetitive DNA Sequences in the genome of Escherichia Coli (E. coli) which could be read as 5’-3’ & 3’-5’ (known as Palindromic Repeats)  [1]. In between these palindromic repeats, there were unique sequences that were referred to as “spacers” [2].  This loci or region of the chromosome was named by scientists as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). At the time, the significance and biological function of CRISPR loci regions were not well understood, but these CRISPR loci were used to genotype various strains of bacteria since these loci had unique variations in different strains of the same species of bacteria [3].  In 1995, Francisco Mojica discovered CRISPR loci in the archaeal genome of Haloferax mediterranei, suggesting their importance in bacterial and archaeal immune systems [4]. Mojica and other labs hypothesized that these loci contained fragments of foreign DNA which is used as a defense mechanism. 

Mechanism of CRISPR-CAS9

When viral DNA is inserted to host bacteria, the host bacteria uses the CAS1 and CAS2 enzyme complex to identify the protospacer from the viral DNA and remove the protospacer upstream of the Protospacer Adjacent Motif (5’-PAM-3’) [5][6]. The protospacer is inserted in the front of the Bacterial CRISPR array, specifically the 5’ end of the repeat, which then pushes the spacer further towards the 5’ end. The initial protospacer from the viral DNA now becomes integrated within the host bacterial CRISPR array [7]. If many different viruses have infected the host bacterium, it may have a long CRISPR Array with many spaces and repeats. Once the host bacterium detects a viral infection, RNA Polymerase will transcribe the entire host bacteria CRISPR Array into single RNA molecules containing repeats & spacer regions called pre-crRNA [8]. Unprocessed trarRNA, which is transcribed from a different region of the host genome, has regions complementary to the regions in crRNA and will bind with the pre-crRNA via hydrogen bonding with the repeat regions [9]. RNAase is an enzyme that cuts repeat regions of the pre-crRNA and what’s left is a single polymer of RNA nucleotides from the repeats & spacers and another single polymer of RNA nucleotides from the unprocessed tracrRNA [10]. These two polymers are referred to as a complex called cr:tracrRNA [11] [12]. Cas9 enzyme binds with this complex and is formed into a guide RNA (gRNA) [13]. The Cas9 consists of 2 essential components: The PAM interacting domain (PI) and the HNC & RuvC nuclease domains. The PI of cas9 can identify the PAM of a viral DNA [14]. The HNH & RuvC nuclease domains can cut DNA [15]. The crRNA carries the sequence information that matches the target DNA and the tracrRNA helps guide the cas9 protein to the target DNA. Cas9 identifies the PAM region of viral DNA and upon detection, cas9 will open the DNA and determine if the gRNA(cr:trarRNA) is complementary to the sequence of bases upstream of PAM on the opposite strand [16].  If the sequence is complementary, cas9 cuts the DNA on both strands from the upstream of PAM, causing a double-strand break (DSB) [17]. The DSB of viral DNA renders it inactive. The final result of CRISPR gene editing is not limited to DSB, this editing tool has been developed for many practical applications. One example is the use of CRISPR dead-endonuclease CAS9 (dCas9) to avoid DNA cleaving of the target gene, rather the Cas9 enzyme is equipped with an activator protein (CRISPRa) or inhibitor (CRISPRi) to regulate the expression of a target gene [18]. 

Practical Use of CRISPR Technology on Eukaryotes

In 2012, Dr. Jennifer Doudna, a molecular biologist from the University of California, Berkeley along with French microbiologist Dr. Emmanuelle Charpentier, were the first to propose that the bacterial CRISPR-Cas9 system can be used for genome editing in humans and other animal species and they received the Nobel Prize in Chemistry for their work in 2020 [19] [20]. They also introduced a refined and simpler version of the CRISPR-Cas9 systems which used RNA that was synthesized in the laboratory and used a single RNA oligo [21]. As mentioned before, the CRISPR system in bacterial cells uses gRNA which has two polymers of RNA (crRNA and tracrRNA) and CRISPR genome editing requires additional procedures in the laboratory to hybridize the two polymers. The introduction of a single RNA oligo referred to as Single-Guide RNA (sgRNA) simplified the experimental design process as researchers only need to synthesize one molecule of RNA, otherwise, researchers would synthesize crRNA and tracrRNA separately in addition to the hybridization process, adding a layer of complexity and variability in experiments. It was later demonstrated that the sgRNA and Cas9 complex was able to determine any sequence of about 20 base pairs as a target for editing and cleaving DNA [22]. For the practical use of CRISPR for gene editing in Eukaryotic Organisms such as humans, a proper sgRNA must be synthesized with the complementary sequence of the target gene, and inserted into a vehicle mechanism which can be either plasmid, RNP, or virus vectors in addition to the Cas9 protein sourced from a bacteria [23] [24]. CRISPR-Cas9 can be used in humans in a multitude of different ways. If a particular sequence of DNA is responsible for a genetic condition, a sgRNA must be developed complementary to that sequence of DNA. Upon recognition from the PI region of the Cas9 protein, the DSB of the sequence can knock out that gene. Unlike prokaryotes, eukaryotes have a DNA repair mechanism that can repair the DNA after DSB from Cas9 [25]. One example of a DNA repair mechanism is Non-homologous End Joining (NHEJ) in which Ku 70/80 protein localizes towards cleaved DNA ends and binds with DNA Protein Kinase Catalytic Subunits to initiate linkage between the cleaved DNA strands [26]. DNA Ligase 4 makes a sugar-phosphate bond called phosphodiester bonds between 2 nucleotides, resulting in ligated ends [27]. This DNA Repair system is error-prone as nucleotides can be added or removed in the joins resulting in indel mutations that disrupt the targeted gene, leading to a gene knock-out [28]. Researchers also developed a way to insert genes by manipulating a natural repair mechanism called Homology Directed Repair. This repair mechanism requires a homologous piece of DNA that acts as a repair template [29]. The repair template is synthesized in the laboratory and has a sequence of DNA added between two homology arms [30]. Cas9 introduces a DSB in the reference DNA and exonucleases trim the broken DNA ends to generate single-stranded overhangs [31]. Rad51 protein, a recombinase protein, facilitates the exchange of DNA strands between the compromised reference DNA and the homologous repair template DNA, knocking-in the intended sequence insertion to the reference DNA. [32] [33] [34]. 

Current Research and Clinical Trials

CRISPR-Cas9 has undergone clinical trials for diseases for the past decade. In March 2020, researchers conducted an in-vivo CRISPR-Cas9 treatment for Leber congenital amaurosis type 10 (LCA10), a condition that affects photoreceptors in the retina, which is caused by a deep intronic mutation in the CEP290 gene [35] [36] . Intronic mutations result in the inclusion of cryptic exons which spliceosomes cannot remove [37]. This can result in the production of abnormal proteins, affecting normal cellular functions. Doctors injected a gene-editing drug into the eye near the retina where there are photoreceptor cells. The drug contained an adeno-associated virus (AAV) vector that contained the CRISPR-CAS9 complex and targeted the CEP290 gene [38]. One of the patients who participated in the study was Carlene Knight. In an interview with NPR, Knight stated the obstacles she faced in daily life before the gene editing treatment “I was bumping into the cubicles and scaring people that were sitting at them” (Stein, 2021)[39]. Knight achieved great results after the treatment and her life became more convenient. For example, she mentioned that she dropped a fork by accident and was appreciative of how she didn’t have to blindly find it when she could simply look for it “I just leaned down to pick it up and didn’t know where it was and just saw it on the floor. It’s very cool”(Stein, 2021). She also mentioned how colors appear much more prominent “I’ve always loved colors. Since I was a kid it’s one of those things I could enjoy with just a small amount of vision. But now I realize how much brighter they were as a kid because I can see them a lot more brilliantly now”(Stein, 2021). Another patient in the experiment reported better visibility of their peripheral vision along with viewing more color. It is important to note that only these 2 out of 7 patients have shown subtle improvements, the rest have shown no results. Mark Pennesi, one of the researchers conducting the treatment, has no explanation as to why the treatment demonstrated no results on the other patients. 

There is promising use of CRISPR technology on patients with sickle-cell disease. Sickle cell disease is a genetic condition in which defective hemoglobin causes red blood cells (RBC) to be sickle-shaped, causing blockage in arteries and can often lead to stroke [40]. To reduce complications of sickle cell disease, researchers have looked into inducing another type of hemoglobin in the body called fetal hemoglobin (HbF). Numerous studies suggest that sickle-cell patients with high HbF count in RBCs report fewer complications as opposed to patients with fewer HbF count [41].  Within a person’s lifetime, HbF transitions to HbA as the BCL11A inhibits the production of HbF in stem cells [42]. By targeting & inactivating the BCL11A gene with CRISPR in stem cells, new RBCs will now have HbF [43]. Victoria Gray, a sickle cell patient, has undergone this gene-editing procedure and the results are amazing. Gray’s fetal hemoglobin levels have increased significantly with reports citing 99.7% of her RBC containing fetal hemoglobin. Gray has mentioned how her life became more convenient after treatment, “Since my treatment, I’ve been able to do everything for myself, everything for my kids. And so it’s been a joy not only for me but for the people around me that’s in my life” (Stein, 2020) [44]. Although the treatment was able to induce physiological change, the long-term risks and implications are unknown as this treatment is fairly new. 

Ethical Considerations

The ethical considerations behind CRISPR-Cas9 gene editing are complicated and there needs to be a balance between the benefits of treating diseases in post-natal patients using CRISPR and the consequences associated with editing germline cells. At a meeting of the European Society of Human Reproduction and Embryology, Dr.Nada Kubikova from the University of Oxford expressed concerns over the use of CRISPR-Cas9 on germline cells (human embryos), “We have found that the DNA of embryo cells can be targeted with high efficiency, but unfortunately this rarely leads to the sort of changes needed to correct a defective gene. More often, the strand of DNA is permanently broken, which could potentially lead to additional genetic abnormalities in the embryo” [45]. The human embryo does not have a sufficient DNA repair mechanism needed to repair a DSB from Cas9 and numerous studies demonstrated significant DNA damage that is not reversible. Kubikova experimented on 84 embryos with 55 embryos serving as control and 33 embryos undergoing DSB using CRISPR-Cas9. In total, 53 DSBs’ were made, and “Of these, 51% (27) were repaired by NHEJ, 9% (5) repaired by HDR and 40% (21) remained unresolved, the persistent breakage eventually causing segmental aneuploidy with a breakpoint at the targeted side” [46]. The unrepaired breaks can cause significant abnormalities if the affected embryos are transferred to a uterus to become a fetus. The embryos undergoing NHEJ would be introduced to new mutation as nucleotides are inserted/deleted in between the breaks. Only 9% of embryos were able to effectively fix the DSB using HDR, and this infrequency poses a concern over CRISPR’s use on embryos.  The current research on CRISPR previously mentioned in this article has been conducted on post-natal individuals but altering the genome at the first stage of development will change every cell as the embryo progresses, and this germline alteration will be passed on to future generations and can have unintended consequences for human evolution. Dr. Greg Licholai, a biotech entrepreneur, expresses great concerns on passing on manipulated genes to subsequent generations, “That’s probably the biggest fear of CRISPR. Humans manipulating the genetic code, and those manipulations get passed on generation to generation to generation. We think we know what we’re doing, we think we’re measuring exactly what changes we’re doing to the genes, but there’s always the possibility that either we miss something or our technology can’t pick up on other changes that have been made that haven’t been directed by us. And the fear then is that those changes lead to antibiotic resistance or other mutations that go out into the population and would be very difficult to control. Basically creating incurable diseases or other potential mutations that we wouldn’t really have control over”(Licholai, 2018) [47].  In the context of post-natal patients, the ethical concerns focus primarily on ensuring the safety, efficacy, and consent of patients undergoing CRISPR. Some studies challenge the safety of CRISPR mentioning that it can unintentionally target pleiotropic genes responsible for the expression of two or more traits. 

Another huge concern is the potential for unintended off-target effects, where CRISPR-Cas9 may inadvertently modify genes other than the targeted ones [48]. This issue is critical due to the complexity of the human genome and the interplay of various genes in regulating various physiological processes. The accuracy of CRISPR-Cas9 targeting specific genes is currently a subject of ongoing research and the consequences from unintended alterations can have a huge impact on patients undergoing gene editing treatment. 

Another ethical concern involves the equitable accessibility to CRISPR-based treatments. Currently, patients can only access CRISPR-based treatments through clinical trials since the therapy is relatively new and considered experimental. Although the pricing of CRISPR is not clear as of yet, the pricing can be inferred based on previous non-CRISPR gene therapies. For example, Sarepta’s drug, which is a gene-therapeutic drug for the treatment of  Duchenne muscular dystrophy, was priced at $750,000 annually [49]. Insurers were reluctant to pay this hefty price tag. If CRISPR gene editing becomes widely available to the general public, there is a risk of creating genetic disparities among populations from different socio-economic groups. Ensuring fair distribution of this technology in different communities is crucial to avoid further health disparities. 

In exploring these ethical challenges, researchers, policymakers, and society at large need to engage in an open and inclusive dialogue on finding a balance between the benefits of CRISPR-Cas9 in treating genetic diseases and the ethical considerations behind germline editing. 

Conclusion

  In conclusion, the ethical implication behind CRISPR-Cas9 gene editing is a complicated issue. The technology is promising for treating many genetic diseases but challenges arise when editing germline cells. Dr. Nada Kubikova’s research underscores the difficulty in achieving precise edits in addition to the risk of potential permanent DNA breaks leading to genetic abnormalities in embryos. This can lead to disastrous genetic consequences for the subsequent generations. As CRISPR advances, it’s important to have a strong ethical laws and open discussions to make sure this gene-editing tech is used responsibly.

References

[1] Nakata, A., Amemura, M., & Makino, K. (1989). Unusual nucleotide arrangement with repeated sequences in the Escherichia coli K-12 chromosome. Journal of Bacteriology, 171(6), 3553–3556. https://doi.org/10.1128/jb.171.6.3553-3556.1989

[2] CRISPR: A Game-Changing Genetic Engineering Technique.” Science in the News, 31 July 2014, https://sitn.hms.harvard.edu/flash/2014/crispr-a-game-changing-genetic-engineering-technique/

. 

[3] Gostimskaya, Irina. “CRISPR-Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing.” Biochemistry. Biokhimiia, U.S. National Library of Medicine, Aug. 2022, www.ncbi.nlm.nih.gov/pmc/articles/PMC9377665/

[4] Mojica, Francisco J.M., et al. “Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements – Journal of Molecular Evolution.” SpringerLink, Springer-Verlag, link.springer.com/article/10.1007/s00239-004-0046-3. Accessed 14 Dec. 2023. 

[5] Dillard, K. E., Brown, M. W., Johnson, N. V., Xiao, Y., Dolan, A., Hernandez, E., … Finkelstein, I. J. (2018). Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex. Cell, 175(4), 934-946.e15. https://doi.org/10.1016/j.cell.2018.09.039

[6] Zhang, J.-H., Adikaram, P., Pandey, M., Genis, A., & Simonds, W. F. (2016). Optimization of genome editing through CRISPR-Cas9 engineering. Bioengineered, 7(3), 166–174. https://doi.org/10.1080/21655979.2016.1189039

[7] Hille, F., & Charpentier, E. (2016). CRISPR-Cas: biology, mechanisms and relevance. Philosophical Transactions of the Royal Society B: Biological Sciences, 371(1707), 20150496. https://doi.org/10.1098/rstb.2015.0496

[8] Karvelis, T., Gasiunas, G., Miksys, A., Barrangou, R., Horvath, P., & Siksnys, V. (2013). crRNA and tracrRNA guide Cas9-mediated DNA interference inStreptococcus thermophilus. RNA Biology, 10(5), 841–851. https://doi.org/10.4161/rna.24203

[9] Trans-Activating crRNA – an overview | ScienceDirect Topics. (n.d.). Retrieved from www.sciencedirect.com website: https://www.sciencedirect.com/topics/neuroscience/trans-activating-crrna#:~:text=The%20secondary%20structure%20on%20the

[10] Xu, T., Li, Y., Van, J. D., He, Z., & Zhou, J. (2014). Cas9-Based Tools for Targeted Genome Editing and Transcriptional Control. 80(5), 1544–1552. https://doi.org/10.1128/aem.03786-13

[11] Ross-Thriepland, D., Bornot, A., Butler, L., Desai, A., Jaiswal, H., Peel, S., … Gianni, D. (2020). Arrayed CRISPR Screening Identifies Novel Targets That Enhance the Productive Delivery of mRNA by MC3-Based Lipid Nanoparticles. SLAS Discovery, 25(6), 605–617. https://doi.org/10.1177/2472555220925770

[12] Berggren, S., Enoksson, I., Stens, C., Lars-Göran, A., & Mårtensson. (n.d.). The CRISPR-Cas system Can CRISPR bring about a brighter future? An extensive and up-to-date CRISPR overview, focusing on the CRISPR-Cas9 technology including a suggestion of a CRISPR-Cas9 laboratory assignment for University students. Retrieved from https://www.diva-portal.org/smash/get/diva2:1510711/FULLTEXT01.pdf

[13] Similar editing efficiency with 2-part and single guide RNA. (n.d.). Retrieved from Integrated DNA Technologies website: https://www.idtdna.com/pages/education/decoded/article/a-high-degree-of-similarity-in-crispr-cas9-editing-efficiency-is-found-between-2-part-guide-rnas-and-single-guide-rnas

[14] Xie, L., Hu, Y., Li, L., Jiang, L., Jiao, Y., Wang, Y., … Yao, S. (2022). Expanding PAM recognition and enhancing base editing activity of Cas9 variants with non‐PI domain mutations derived from xCas9. The FEBS Journal, 289(19), 5899–5913. https://doi.org/10.1111/febs.16457

[15] Babu, K., Kathiresan, V., Kumari, P., Newsom, S., Parameshwaran, H. P., Chen, X., … Rajan, R. (2021). Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence. Biochemistry, 60(49), 3783–3800. https://doi.org/10.1021/acs.biochem.1c00354

[16] Gleditzsch, D., Pausch, P., Müller-Esparza, H., Özcan, A., Guo, X., Bange, G., & Randau, L. (2019). PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures. RNA Biology, 16(4), 504–517. https://doi.org/10.1080/15476286.2018.1504546

[17] Wu, X., Kriz, A. J., & Sharp, P. A. (2014). Target specificity of the CRISPR-Cas9 system. Quantitative Biology, 2(2), 59–70. https://doi.org/10.1007/s40484-014-0030-x

[18] Karlson, C. K. S., Mohd-Noor, S. N., Nolte, N., & Tan, B. C. (2021). CRISPR/dCas9-Based Systems: Mechanisms and Applications in Plant Sciences. Plants, 10(10), 2055. https://doi.org/10.3390/plants10102055

[19] Zhu, M. (2012). Jennifer Doudna and Emmanuelle Charpentier’s Experiment About the CRISPR/cas 9 System’s Role in Adaptive Bacterial Immunity (2012) | The Embryo Project Encyclopedia. Retrieved from Asu.edu website: https://embryo.asu.edu/pages/jennifer-doudna-and-emmanuelle-charpentiers-experiment-about-crisprcas-9-systems-role-adaptive

[20] Sanders, R., & relations |, M. (2020, October 7). UC Berkeley’s Jennifer Doudna wins 2020 Nobel Prize in Chemistry. Retrieved from Berkeley News website: https://news.berkeley.edu/2020/10/07/jennifer-doudna-wins-2020-nobel-prize-in-chemistry

[21] 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

[22] Yau, E. H., & Rana, T. M. (2018). Next-Generation Sequencing of Genome-Wide CRISPR Screens. Methods in Molecular Biology (Clifton, N.J.), 1712, 203–216. https://doi.org/10.1007/978-1-4939-7514-3_13

[23] Horodecka, K., & Düchler, M. (2021). CRISPR/Cas9: Principle, Applications, and Delivery through Extracellular Vesicles. International Journal of Molecular Sciences, 22(11), 6072. https://doi.org/10.3390/ijms22116072

[24] Zhong, Z., Liu, G., Tang, Z., Xiang, S., Yang, L., Huang, L., … Zhang, Y. (2023). Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system. Nature Communications, 14(1), 6102. https://doi.org/10.1038/s41467-023-41802-9

[25] Lieber, M. R. (2010). The Mechanism of Double-Strand DNA Break Repair by the Nonhomologous DNA End-Joining Pathway. Annual Review of Biochemistry, 79(1), 181–211. https://doi.org/10.1146/annurev.biochem.052308.093131

[26] Zahid, S., Seif El Dahan, M., Iehl, F., Fernandez-Varela, P., Le Du, M.-H., Ropars, V., & Charbonnier, J. B. (2021). The Multifaceted Roles of Ku70/80. International Journal of Molecular Sciences, 22(8), 4134. https://doi.org/10.3390/ijms22084134

[27] Kaminski, A. M., Tumbale, P. P., Schellenberg, M. J., Williams, R. S., Williams, J. G., Kunkel, T. A., … Bebenek, K. (2018). Structures of DNA-bound human ligase IV catalytic core reveal insights into substrate binding and catalysis. Nature Communications, 9(1), 2642. https://doi.org/10.1038/s41467-018-05024-8

[28] Guo, T., Feng, Y.-L., Xiao, J.-J., Liu, Q., Sun, X.-N., Xiang, J.-F., … Xie, A.-Y. (2018). Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing. Genome Biology, 19(1). https://doi.org/10.1186/s13059-018-1518-x

[29] Bloh, K., & Rivera-Torres, N. (2021). A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity. International Journal of Molecular Sciences, 22(8), 3834. https://doi.org/10.3390/ijms22083834

[30] Schubert, M. S., Thommandru, B., Woodley, J., Turk, R., Yan, S., Kurgan, G., … Rettig, G. R. (2021). Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair. Scientific Reports, 11(1). https://doi.org/10.1038/s41598-021-98965-y

[31] Zhao, F., Kim, W., Kloeber, J. A., & Lou, Z. (2020). DNA end resection and its role in DNA replication and DSB repair choice in mammalian cells. Experimental & Molecular Medicine, 52(10), 1705–1714. https://doi.org/10.1038/s12276-020-00519-1

[32] Chen, J., Morrical, M. D., Donigan, K. A., Weidhaas, J. B., Sweasy, J. B., Averill, A. M., … Morrical, S. W. (2014). Tumor-associated mutations in a conserved structural motif alter physical and biochemical properties of human RAD51 recombinase. Nucleic Acids Research, 43(2), 1098–1111. https://doi.org/10.1093/nar/gku1337

[33] Lin, Z., Kong, H., Nei, M., & Ma, H. (2006). Origins and evolution of the recA/RAD51 gene family: Evidence for ancient gene duplication and endosymbiotic gene transfer. Proceedings of the National Academy of Sciences, 103(27), 10328–10333. https://doi.org/10.1073/pnas.0604232103

[34] Gallagher, D. N., Pham, N., Tsai, A. M., Janto, A. N., Choi, J., Ira, G., & Haber, J. E. (2020). A Rad51-independent pathway promotes single-strand template repair in gene editing. PLOS Genetics, 16(10), e1008689. https://doi.org/10.1371/journal.pgen.1008689

[35] Groundbreaking CRISPR treatment for blindness only works for subset of patients. (n.d.). Retrieved from www.science.org website: https://www.science.org/content/article/groundbreaking-crispr-treatment-blindness-only-works-subset-patients

[36] Dulla, K., Aguila, M., Lane, A., Jovanovic, K., Parfitt, D. A., Schulkens, I., … Cheetham, M. E. (2018). Splice-Modulating Oligonucleotide QR-110 Restores CEP290 mRNA and Function in Human c.2991+1655A>G LCA10 Models. Molecular Therapy – Nucleic Acids, 12, 730–740. https://doi.org/10.1016/j.omtn.2018.07.010

[37] Anna, A., & Monika, G. (2018). Splicing mutations in human genetic disorders: examples, detection, and confirmation. Journal of Applied Genetics, 59(3), 253–268. https://doi.org/10.1007/s13353-018-0444-7

[38] ​​Henderson, H. (2022, March 29). CRISPR Clinical Trials: A 2022 Update. Retrieved from Innovative Genomics Institute (IGI) website: https://innovativegenomics.org/news/crispr-clinical-trials-2022/

[39] Stein, R. (2021, September 29). A Gene-Editing Experiment Let These Patients With Vision Loss See Color Again. NPR.org. Retrieved from https://www.npr.org/sections/health-shots/2021/09/29/1040879179/vision-loss-crispr-treatment

[40] CDC. (2022). What Is Sickle Cell Disease? Retrieved from Centers for Disease Control and Prevention website: https://www.cdc.gov/ncbddd/sicklecell/facts.html

[41] Antwi-Boasiako, C., Frimpong, E., Ababio, G., Dzudzor, B., Ekem, I., Gyan, B., … Antwi, D. (2015). Sickle cell disease: reappraisal of the role of foetal haemoglobin levels in the frequency of vaso-occlusive crisis. Ghana Medical Journal, 49(2), 102. https://doi.org/10.4314/gmj.v49i2.7

[42] ​​Sankaran, V. G., & Orkin, S. H. (2012). The Switch from Fetal to Adult Hemoglobin. Cold Spring Harbor Perspectives in Medicine, 3(1), a011643–a011643. https://doi.org/10.1101/cshperspect.a011643

[43] Khosravi, M. A., Abbasalipour, M., Concordet, J.-P., Berg, J. V., Zeinali, S., Arashkia, A., … Karimipoor, M. (2019). Targeted deletion of BCL11A gene by CRISPR-Cas9 system for fetal hemoglobin reactivation: A promising approach for gene therapy of beta thalassemia disease. European Journal of Pharmacology, 854, 398–405. https://doi.org/10.1016/j.ejphar.2019.04.042

[44] Stein, R. (2020, June 23). A Year In, 1st Patient To Get Gene Editing For Sickle Cell Disease Is Thriving. Retrieved from NPR.org website: https://www.npr.org/sections/health-shots/2020/06/23/877543610/a-year-in-1st-patient-to-get-gene-editing-for-sickle-cell-disease-is-thriving

[45] Cooke, E. (2023, June 29). CRISPR gene editing on human embryos may have dangerous consequences, says new study. Retrieved from Clinical Trials Arena website: https://www.clinicaltrialsarena.com/news/crispr-gene-editing-on-human-embryos-may-have-dangerous-consequences-says-new-study/?cf-view

[46] N Kubikova, M Esbert, Titus, S., C Coudereau, M Savash, Fagan, J., … Wells, D. (2023). O-075 Deficiency of DNA double-strand break repair in human preimplantation embryos revealed by CRISPR-Cas9. 38(Supplement_1). https://doi.org/10.1093/humrep/dead093.089

[47] Licholai, G. (2018, August 21). Is CRISPR Worth the Risk? Retrieved from Yale Insights website: https://insights.som.yale.edu/insights/is-crispr-worth-the-risk

[48] Guo, C., Ma, X., Gao, F., & Guo, Y. (2023). Off-target effects in CRISPR/Cas9 gene editing. Frontiers in Bioengineering and Biotechnology, 11(1143157). https://doi.org/10.3389/fbioe.2023.1143157

[49] Sherkow, J. S. (2017). CRISPR, Patents, and the Public Health. The Yale Journal of Biology and Medicine, 90(4), 667–672. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733839/