¹ Shenghua Zizhu Academy, No.155, Tanjiatang Road, Minhang District, Shanghai 20024, China;

² Diamond Bar High School, 21400 Pathfinder Rd, Diamond Bar, CA 91765, USA

Corresponding Author: Jasmine Shi, Diamond Bar High School, 21400 Pathfinder Rd, Diamond Bar, CA 91765, E-mail: jjasmineshi123@hotmail.com.

Running title: ETHICAL CONCERNS OF CRISPR/CAS9-BASED GERMLINE GENOME EDITING

	ABSTRACT
	INTRODUCTION
	MECHANISM OF THE CRISPR/CAS9
	THERAPEUTIC BENEFITS OF CRISPR/CAS9-BASED GERMLINE GENOME EDITING
	SAFETY AND ETHNICAL CONCERNS OF THE CRISPR/CAS9-BASED GERMLINE GENOME EDITING
	REGULATIONS OF THE CRISPR/CAS9-BASED GERMLINE GENOME EDITING
	CONCLUSION
	ACKNOWLEDGMENTS
	CONFLICT OF INTEREST
	REFERENCES

	ABSTRACT

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 adapted from natural immune defense systems of bacteria and archaea can edit genes in numerous organisms including humans. This has revolutionized genetic engineering and can help better understand genetic diseases as well as potentially correct causative mutations. Current CRISPR/Cas9-based genome editing is focused on either somatic cells or germline cells. In contrast to somatic cell genome editing, germline genome editing raises significant ethical issues as the genomic modifications in those reproductive cells by CRISPR/Cas9 can potentially be passed on to future generations. In this article, we focus on discussion of safety and ethical issues of CRISPR/Cas9-based germline genome editing in humans from several aspects including off-targets, Immunogenicity, autonomy, religion and eugenics.

	INTRODUCTION

   CRISPR/Cas9 is an adaptive immune system of bacteria and archaea to protect themselves from virus invasion by degrading foreign virial genetic elements (1, 2). In 2013, Jennifer Doudna and other scientists proved that the CRISPR/Cas9 system can be adapted for genome editing in many organisms including humans, and this significant discovery led to the Nobel Prize award to Jennifer Doudna and Emmanuelle Charpentier in 2020 (3-5). The CRISPR/Cas9 technique makes possible to analyze gene functions in cells, study disease progression, and potentially correct genetic mutations accounting for diseases (6). Based on the type of cells being edited, the CRISPR/Cas9 genome-editing can be classified into somatic cell genome editing and germline genome editing. Somatic cell genome editing refers to modifications of the genomes of body tissue cells, such as fibroblasts and muscle cells, to treat or cure diseases. As the somatic cell genome editing focuses on non-reproductive cells, the effects of genome changes are restricted to the treated individual and would not be inherited by future generations. Therefore, the CRISP/Cas9-based somatic cell genome editing is generally morally acceptable. In 2018, the National Institute of Health (NIH) launched the Somatic Cell Genome Editing program with aims to develop high-quality genome editing tools to reduce the burden of common and rare diseases caused by genetic changes (7). To date, all the clinical trials of CRISP/Cas9-based therapies are the somatic cell genome editing.

   In contrast to somatic cell genome editing, the germline genome editing conducted on embryos, zygotes, or gametes could lead to heritable changes to future generations, causing unpredictable dangers. Therefore, human germline genome editing has raised significant ethical issues, particularly after the birth of the world’s first CRISPR/Cas9 germline-edited human babies with the intention to create immunity to HIV infection in 2018 (8). In this article, we focus on safety and ethical discussion of CRISPR/Cas9-based germline genome editing in humans and potential future perspectives.

	MECHANISM OF THE CRISPR/CAS9

   The CRISPR/Cas9 system has attracted significant attention in genome editing due to its efficiency, affordability and precision. This system is composed of two components, Cas9 endonuclease and a short guide RNA (gRNA) comprising a target-specific CRISPR RNA (crRNA) and a trans-activating RNA (tracrRNA) (2, 9). When introduced into cells, the gRNA can recognize the targeted DNA sequence by nucleotide complementary base pairing with the Cas9 binding simultaneously with the aid of a three- nucleotide Protospacer-Adjacent Motif (PAM) on the 5’ end of the target strand (10) (Figure 1). Then the Cas9 endonuclease precisely cuts DNA at the target sites and generates specific double-stranded breaks (DSBs), followed by cellular DNA damage response machinery for repair via two mechanisms: the error-prone non-homologous end joining (NHEJ) and the homology-directed repair (HDR) (Figure 1). NHEJ mechanism could induce insertion, deletion or frameshift at the targeted DNA site, while HDR mechanism can lead to precise nucleotide edits by using DNA repair templates (Figure 1) (2, 9, 11). The efficiency, specificity, ease of use and inexpensiveness of the CRISP/Cas9 system make it an extremely powerful tool for research and clinical applications.

View larger version : [in a new window] Figure 1. Scheme of CRISPR/Cas9-mediated genome editing. Cas9 nuclease cleaves double-stranded DNA directed by a single guide RNA (sgRNA) which is complementary to the target DNA sequence upon the presence of a protospacer-adjacent motif (PAM). The resultant double-strand breaks (DSBs) then trigger the cellular machinery repairing system via either nonhomologous end-joining (NHEJ) or by homology directed repair (HDR) with a donor template.

	THERAPEUTIC BENEFITS OF CRISPR/CAS9-BASED GERMLINE GENOME EDITING

   Heritable diseases collectively affect 5%-7% of the human population with an estimated 10,000 diseases resulted from mutations of single genes (12). The advent of CRISPR/Cas9 provides a great potential to correct the mutations in the germline and create embryos free of disease-causing mutations, which could avoid the diseased gene to be passed on to the future generations. For instance, cystic fibrosis is an inherited genetic disorder caused by homozygous mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which affects the function of the lungs, pancreas, and other organs. Adult cystic fibrosis patients who want to raise a family may wish to avoid passing on the disease to their children, and such a requirement could be met by the CRISPR/Cas9 germline genome editing.

   The CRISPR/Cas9 germline genome editing can also apply to diseases caused by out-of-frame gene mutations. For example, Duchenne muscular dystrophy (DMD), an X-linked recessive neuromuscular disorder, affects 1 out of 3500 males. The patients generally develop muscular dystrophies, loss of ambulation and cardiomyopathy, and die in their twenties. The most common cause of DMD is out-of-frame mutations in the dystrophin gene, and currently no specific treatments are available. CRISPR/Cas9 germline genome editing is a promising therapeutic tool for DMD by permanently correcting DMD mutations and restoring the production of functional dystrophin (13).

   In addition to the diseases resulted from the single gene mutations and the out-of-frame gene mutations, aneuploidy-related genetic disorders may also be treated by CRISPR/Cas9 germline genome editing in the future. Aneuploidy is the presence of an extra or a missing chromosome in cells which leads to spontaneous abortions and developmental disabilities. The common aneuploidy disorders include down syndrome (an extra copy of chromosome 21), Edwards syndrome (an extra copy of chromosome 18) and Patau syndrome (an extra copy of chromosome 13) that affect millions of people worldwide. The CRISPR /cas9 based-germline genome editing provides a great tool to correct chromosome abnormality, which could otherwise pass on to the patients’ offspring. Zuo et al reported that the application of CRISPR/Cas9 selectively eliminated an extra copy of chromosome 21 in human induced pluripotent stem cells on the targeted chromosome (14).

   In the long term, the germline genome editing would reduce targeted heritable diseases prevalence or even eliminate the heritable diseases in human population. For this reason, Christopher Gyngell and colleagues believe that human germline genome editing is rather than being merely ’morally permissible’, but a “moral imperative”(15).

	SAFETY AND ETHNICAL CONCERNS OF THE CRISPR/CAS9-BASED GERMLINE GENOME EDITING

   Off-target issues of the CRISP/Cas9 technology

   Although the targeting specificity of Cas9 is controlled by an approximately 20-nucleotides’ gRNA complementary to the target DNA sequence with a PAM site adjacent to the target sequence in the genome, off-target cleavages could still occur on in the genome. Some studies have shown that different gRNA structures can affect the cleavage of off-target sites. Cas9 can recognize not only 5’-NGG-3′ PAM sequence but also other PAM sequences such as 5’-NAG-3′ and 5’-NGA-3′, leading to unintended genomic alterations that may have detrimental effects on cellular function (16-18). Cong et al have demonstrated that some gRNAs containing up to five mismatches with target sequences induced CRISPR-mediated cleavage (3). Pattanayak et al also have shown that the cleavage events occurred at off-target sequences with up to seven mismatches against treated gRNAs, indicating that incomplete complementarity between the gRNA and the target DNA still induces CRISPR/Cas9-mediated edits (19). Particularly, in a study in 2015 to edit HBB gene encoding the β-globin protein in human embryos, Dr. Huang found that the off-target mutation rate was much higher than those observed in gene-editing studies of mouse embryos or human adult cells (20). The off-target cleavages of the CRISPR/Cas9 system have raised significant concerns for the applications of the Crispr/Cas9 in the human germline genome editing as it may introduce unintended gene mutations which can be passed on to the future generations.

   To minimize the off-target effects of the CRISPR/Cas9 genome editing, several strategies have been developed. Firstly, optimizing gRNA designs could maximize specificity and minimize the off-target effects of the CRISPR/Cas9 by using bioinformatics tools to predict potential off-target sites (21, 22); Secondly, developing high-fidelity Cas9 variants, such as SpCas9-HF1 or eSpCas9, were reported with the reduced off-target effects compared to the wild-type Cas9 (23, 24); Thirdly, using off-target detection methods, such as GUIDE-seq or CIRCLE-seq, can identify and quantify off-target effects of CRISPR/Cas9 (25, 26).

   Though those strategies could reduce the off-target effects of the CRISPR/Cas9, a complete elimination of its off-target effects is difficult to achieve presently. Thus, the current CRISPR/Cas9 based-germline genome editing could still produce unpredictable risks of creating novel genome mutations which can impact future generations.

   Immunogenicity of CRISPR/Cas9

   The most commonly used Cas9 orthologues are derived Staphylococcus aureus or Streptococcus pyogenes, two bacterial species which infect the human population at high frequencies. Therefore, humans could have already harbored pre-existing adaptive immune responses to the Cas9 orthologs derived from the bacteria, leading to the safe concerns and efficacious use of CRISPR/Cas9 when treating patients. In 2019, Charlesworth et al reported the detection of specific antibodies and T cells in human cord and peripheral blood against Cas9 derived from both Staphylococcus aureus and Streptococcus pyogenes by using an enzyme-linked immunosorbent assay (27). Their study confirms that there are pre-existing adaptive immunity responses against the CRISPR/Cas9 system in humans, and activation of the adaptive immunity by the CRISPR/Cas9 technology could promote systemic inflammation or cause devastating toxicity to patients (27). Other than the effects on adaptive immunity, gRNAs in the Crispr/Cas9 system may induce innate immune responses in human cells as well. Kim et al reported that gRNAs prepared with a 5′-triphosphate group (5′-ppp gRNAs) via in vitro transcription from DNA trigger innate immune responses to gRNAs in human and murine cells, leading to cytotoxicity (28).

   As the CRISPR/Cas9 system may induce activation of both the adaptive and innate immunity responses in humans which further complicate the clinical utilization of this technology, several strategies have been developed to minimize or eliminate the induced-immunity responses are needed in the CRISPR/Cas9-based germline genome editing. Ferdosi et al reported that modified Cas9 protein by gene mutations can eliminate immunodominant epitopes without loss of its function and specificity (29). In addition, Cas9 orthologs of non-pathogenic bacteria from extreme habitats without any prior exposure to humans could circumvent the problem of pre-existing antibodies and antigen-specific T cells in humans. For instance, Cas9 from Geobacillus stearothermophilus was found to be highly stable in human plasma (30).

   Autonomy: Lack of Informed Consent by the Child Affected by the Edits

   In human germline genome editing, it is the parent(s) who actually make the decision on behalf of their future children. Therefore, some argued against human germline genome editing as it lacks the consent of the future children. In 2015, Francis S. Collins, Ph.D., director of the NIH, stated that “ethical issues presented by altering the germline in a way that affects the next generation without their consents (31). This argument has been supported by many scientists. For instance, Mintz noted that the decision taken by the parents could affect its future autonomy though the embryo does not have the capacity for autonomous decision-making at the time of the germline genome editing (32). While John Harris, professor Emeritus of University of Manchester had a different view on it: “consent issues, as we have noted, are irrelevant because consent is never available from the unborn or for things that might affect future generations. We have to address dangers to future generations and to the planet in quite another way; the raising of the issue of consent or its absence in such cases is, to put the point as politely as possible, irrelevant and misleading” (33). In support of John Harris’ view, Gyngell and colleagues illustrated: if the germline genome editing can prevent a future child from suffering cystic fibrosis, this child, once born, can live without the consequences of cystic fibrosis, and the germline genome editing actually promotes the individual’s autonomy (34).

   Religious concerns

   Religions play significant roles in societal values, and religious beliefs may influence perceptions of germline genome editing applications.

   In Islam's point of view, they believe that genomic alterations in the body may have a marked effect on a person's behavior and may leave metaphysical and spiritual imbalances as a result, and everyone has the right to inherit a genome that has not been artificially altered. In addition, they believe that God has created humans in their best form, and Muslims must strive for righteousness and spirituality rather than physical perfection (35). However, some Islam believers think that under certain conditions, gene editing of the human germline would be considered legal for medical purposes (36).

   In Christianity, there are varying opinions on germline genome editing. The Catholic Church has been firmly against germline genome editing. In an interview with EWTN Pro-Life Weekly on June 25, 2020, the president of the National Catholic Bioethics Center, Joseph Meaney (PhD), stated that the Church must stand firm against the unborn being "sacrificed on the altar of scientific research"(37). They believe that germline genome editing is immoral as it manipulates human life at its earliest stages and could lead to unintended consequences for future generations. While some Christians believe that germline genome editing could be acceptable to prevent or cure genetic diseases, and they view germline genome editing as a way to fulfill the Christian duty of caring for the sick and vulnerable and promote human flourishing(38).

   From Buddhism point of view, they believe that human action and freedom are interpreted in relation to morality. A balance between mental and physical health is essential, as illness is believed to be caused by past actions, as in karma (39). They believe that any destructive embryonic experimentation or research activity violates the do-no-harm principle. Moreover, editing of germ cells is morally impermissible because of its negative impact on society and humanity. In general, Buddhism believes that what you cannot control according to your desires is not yours.

   In conclusion, most religions are against human germline editing though some believers of each of these religions may support it.

   Eugenics concerns

   Eugenics refers to the selection of desired heritable traits to improve future generations, and the traits may include physical strength, intelligence, and longevity. The CRISPR/Cas9 germline genome editing has the potential to enhance human characteristics, and create a genetically superior class of humans which leads to various types of injustices as well as social and economic inequalities.

   In sports, there is a large admiration for professional athletes who have superior individual performances. However, there is great contempt for individual athletes who choose to act selfishly for the benefit of themselves such as using performance-enhancing drugs. Similarly, individuals would have an unfair advantage for their superior performances resulting from the application of germline genome editing.

   Human enhancement through the germline genome editing could create or accelerate social and economic inequalities in many aspects. For instance, people will have unequal access to the CRISPR/Cas9 technology for enhancement as only the wealthy may afford it, creating a situation that those who can afford the technology have an unfair advantage over those who cannot. Even if one day, CRISPR/Cas9 germline genome editing for human enhancement becomes widely accessible, it may result in new forms of discrimination. For instance, employers might like to hire only workers with certain enhancements, leading to disadvantage for the workers without them.

	REGULATIONS OF THE CRISPR/CAS9-BASED GERMLINE GENOME EDITING

   In recent years, multiple professional organizations have held discussion meetings and released over 60 statements pertaining to human germline genome editing (40). Most of those statements insisted that human germline genome editing should be banned, whereas some statements indicated that it could be permissible with strict oversight and regulation to ensure safety and ethical considerations are adequately addressed (41, 42). Ethical statements are important, but their impacts may be limited as consequences for violating ethical statements are typically less severe including the loss of funding, publication retraction, and the loss of trust among the scientific community. Recently, many governments have established regulations for the CRISPR/Cas9 germline genome editing, and most of the regulations completely ban human germline genome editing. For example, the Food and Drug Administration (FDA) in the United States has stated that it will not approve any clinical trials involving human germline editing. In the United Kingdom, human germline genome editing is permitted for research purposes only, but not for clinical applications (43).

   Given the fact that countries may have different legal and social considerations on Crispr/Cas9 germline genome editing, it is reasonable for the global community to contemplate implementing supranational regulations. In 2021, the World Health Organization issued the recommendations on Human Genome Editing for the Advancement of Public Health [2021] with the intention to set global standards for governance and oversight of human genome editing (44).

	CONCLUSION

   The advent of the CRISPR/Cas9 technology has revolutionized genome editing, offering the possibility of treating and curing genetic diseases in humans. The CRISPR/Cas9-based somatic cell genome editing is generally morally acceptable as it affects only a subset of cells within a single organ, and its impacts would not be passed on to future generations (7). In contrast, the CRISPR/Cas9-based germline genome editing raises a number of ethical concerns since it involves DNA changes to sperm, eggs, or embryos which affects all of the cells including germ cells in a person, leading to heritable genome changes to future generations.

   One of main ethical concerns in the CRISPR/Cas9-based germline genome editing is safety and unforeseeable consequences including the off-target issue and immunogenicity of the CRISPR/Cas9 which could have serious and irreversible consequences for future generations. Nevertheless, recently many strategies have been developed to minimize the off-target effects of the CRISPR/Cas9 genome editing such as optimizing gRNA designs and developing high-fidelity Cas9 variants (21-24). In addition, studies have shown immunogenicity of the CRISPR/Cas9 could be minimized by modifying Cas9 protein and utilizing Cas9 orthologs isolated from nonpathogenic bacteria (29, 30). As the rapid development of technology, we are optimistic that safety issues associated with CRISPR/cas9 system, such as off-target issue and immunogenicity, will be eventually overcome.

   In addition, autonomy of individuals and the possibility of eugenics are another two ethical concerns for germline genome editing. As CRISPR/Cas9 germline genome editing modifies the genetic material of an individual before they are born, it raises questions about autonomy. Furthermore, germline genome editing could be used to select desirable traits or characteristics, creating a society where certain traits are deemed more valuable than others. This could lead to discrimination against individuals who do not possess these traits and could potentially widen existing societal inequalities.

   As religions play significant roles in societal values, religious beliefs would impact people’s perceptions on CRISPR/Cas9-based germline genome editing. Different religions hold different views on germline genome editing, and the act of genetic genome editing has been interpreted differently in each religion. However, most religions are not very supportive of germline genome editing, which they believe is immoral and can have unpredictable effects on individuals and society.

   Overall, the ethical implications of CRISPR/Cas9-based germline genome editing are complex and multifaceted, and careful consideration and discussion are required among scientists, policymakers, and the general public. Eventually, global uniformity in the regulations and standards would be beneficial as ethical implications of the CRISPR9/Cas9 technology extend beyond national boundaries. The future of germline genome editing in humans remains uncertain, but it is clear that any clinical application of germline genome editing would need to be carefully regulated and evaluated to ensure safety and ethical considerations.

	ACKNOWLEDGMENTS

   I would like to acknowledge my mentor, Dr. Jay Hao, for guiding me through this paper and providing insightful resources and ideas.

	CONFLICT OF INTEREST

   The authors disclose no conflict of interest.

	REFERENCES

1. Barrangou R, Fremaux C, Deveau H, Richards M, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007; 315 (5819): 1709-1712.
2. Jinek M, Chylinski K, Fonfara I, Hauer M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337 (6096): 816-821.
3. Cong L, Ran FA, Cox D, Lin S, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339 (6121): 819-823.
4. Jinek M, East A, Cheng A, Lin S, et al. RNA-programmed genome editing in human cells. Elife. 2013; 2: e00471.
5. Mali P, Yang L, Esvelt KM, Aach J, et al. RNA-guided human genome engineering via Cas9. Science. 2013; 339 (6121): 823-826.
6. Knott GJ, Doudna JA. CRISPR-Cas guides the future of genetic engineering. Science. 2018; 361 (6405): 866-869.
7. Somatic Cell Genome Editing [Available from: https://commonfund.nih.gov/editing. (Access date: March 22, 2023)
8. Greely HT. CRISPR'd babies: human germline genome editing in the 'He Jiankui affair'. J Law Biosci. 2019; 6 (1): 111-183.
9. Karvelis T, Gasiunas G, Miksys A, Barrangou R, et al. crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus. RNA Biol. 2013; 10 (5): 841-851.
10. Asmamaw M, Zawdie B. Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing. Biologics. 2021; 15: 353-361.
11. Komor AC, Badran AH, Liu DR. CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell. 2017; 168 (1-2): 20-36.
12. Kofler N, Kraschel KL. Treatment of heritable diseases using CRISPR: Hopes, fears, and reality. Semin Perinatol. 2018; 42 (8): 515-521.
13. Erkut E, Yokota T. CRISPR Therapeutics for Duchenne Muscular Dystrophy. Int J Mol Sci. 2022; 23 (3).
14. Zuo E, Huo X, Yao X, Hu X, et al. CRISPR/Cas9-mediated targeted chromosome elimination. Genome Biol. 2017; 18 (1): 224.
15. Gyngell C, Bowman-Smart H, Savulescu J. Moral reasons to edit the human genome: picking up from the Nuffield report. J Med Ethics. 2019; 45 (8): 514-523.
16. Alkan F, Wenzel A, Anthon C, Havgaard JH, Gorodkin J. CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters. Genome Biol. 2018; 19 (1): 177.
17. Fu Y, Foden JA, Khayter C, Maeder ML, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013; 31 (9): 822-826.
18. Hsu PD, Scott DA, Weinstein JA, Ran FA, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013; 31 (9): 827-832.
19. Pattanayak V, Lin S, Guilinger JP, Ma E, et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 2013; 31 (9): 839-843.
20. Liang P, Xu Y, Zhang X, Ding C, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015; 6 (5): 363-372.
21. Bae S, Park J, Kim JS. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 2014; 30 (10): 1473-1475.
22. Concordet JP, Haeussler M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 2018; 46 (W1): W242-W245.
23. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016; 529 (7587): 490-495.
24. Slaymaker IM, Gao L, Zetsche B, Scott DA, et al. Rationally engineered Cas9 nucleases with improved specificity. Science. 2016; 351 (6268) :84-88.
25. Tsai SQ, Zheng Z, Nguyen NT, Liebers M, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015; 33 (2): 187-197.
26. Tsai SQ, Nguyen NT, Malagon-Lopez J, Topkar VV, et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat Methods. 2017; 14 (6): 607-614.
27. Charlesworth CT, Deshpande PS, Dever DP, Camarena J, et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat Med. 2019; 25 (2): 249-254.
28. Kim S, Koo T, Jee HG, Cho HY, et al. CRISPR RNAs trigger innate immune responses in human cells. Genome Res. 2018; 28 (3): 367-373.
29. Ferdosi SR, Ewaisha R, Moghadam F, Krishna S, et al. Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes. Nat Commun. 2019; 10 (1): 1842.
30. Harrington LB, Paez-Espino D, Staahl BT, Chen JS, et al. A thermostable Cas9 with increased lifetime in human plasma. Nat Commun. 2017; 8 (1): 1424.
31. National Institutes of Health. Statement on NIH funding of research using gene-editing technologies in human embryos 2015 [Available from: https://www.nih.gov/about-nih/who-we-are/nih-director/statements/statement-nih-funding-research-using-gene-editing-technologies-human-embryos. (Access date: March 12, 2023)
32. Mintz RL, Loike JD, Fischbach RL. Will CRISPR Germline Engineering Close the Door to an Open Future? Sci Eng Ethics. 2019; 25 (5): 1409-1423.
33. Harris J. Germline Modification and the Burden of Human Existence. Camb Q Healthc Ethics. 2016; 25 (1): 6-18.
34. Gyngell C, Douglas T, Savulescu J. The Ethics of Germline Gene Editing. J Appl Philos. 2017; 34 (4): 498-513.
35. Kalidasan V, Das KT. Playing God? Religious Perspectives on Manipulating the Genome. J Relig Health. 2022; 61 (4): 3192-3218.
36. Isa NM, Zulkifli NA, Man S. Islamic Perspectives on CRISPR/Cas9-Mediated Human Germline Gene Editing: A Preliminary Discussion. Sci Eng Ethics. 2020; 26 (1): 309-323.
37. RSS NN. EWTN Pro-Life Weekly: NCBC President Discusses UK Gene Editing Experiment 2020 [Available from: https://www.ncbcenter.org/ncbc-news/ewtn-pro-life-weekly-ncbc-president-discusses-uk-gene-editing-experiment. (Access date: March 20, 2023).
38. Available from: https://www.christianitytoday.com/news/2020/december/pew-survey-global-bioethics-gene-editing-crispr-christians.htm. (Access date: March 20, 2023).
39. Sayadaw VM. The theory of Karma [Available from: https://www.buddhanet.net/e-learning/karma.htm.( Access date: March 12, 2023).
40. Brokowski C. Do CRISPR Germline Ethics Statements Cut It? CRISPR J. 2018; 1 (2): 115-125.
41. Netherlands CoGMatHCot. Editing human DNA: Moral and social implications of germline genetic modification. 2017.
42. Bioethics. TAoMSrttNCo. Genome editing and human reproduction 2017 [Available from: https://acmedsci.ac.uk/file-download/89659337.(Access date: March 25, 2023).
43. United Kingdom: Germline / Embryonic [Available from: https://crispr-gene-editing-regs-tracker.geneticliteracyproject.org/united-kingdom-germline-embryonic/#:~:text=Germline%20gene%20editing%20is%20permitted,for%20reproductive%20purposes%20is%20illegal.( Access date: March 22, 2023)
44. Organization WH. WHO issues new recommendations on human genome editing for the advancement of public health 2021 [Available from: https://www.who.int/news/item/12-07-2021-who-issues-new-recommendations-on-human-genome-editing-for-the-advancement-of-public-health. (Access date: March 25, 2023)

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