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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. Keywords— CRISPR/Cas9, genome editing, gene therapy, sgRNA 1. USING CRISPR-CAS9 FOR THERAPEUTIC PROTEIN PRODUCTION Evidence of a genetic etiology for various illnesses has increased efforts to find remedies that target the defective genes. Specifically, with the completion of the human genome project, scientist have been able to map out the loci of genes implicated in various ailments such as cancers and thus potentiating the use of genetic engineering technologies to edit such defective genes. One of these genome-editing technologies is the CRISPR/Cas9 system, a customizable approach to edit DNA based on the ability of the endonuclease Cas9 to bind and cleave specific nucleotide sequences in the human genome [1]. CRISPR/Cas systems are systems naturally developed in bacteria and archaea that confer immunity to these organisms against invading plasmids and viruses [2]. The adaptive immunity provided by these systems in the prokaryotes arises from the ability of CRISPR RNAs (crRNAs) to target the invading nucleic acids thus leading to the silencing of these invading nucleic acids through their cleavage and integration of the resultant fragments into the CRISPR locus [3]. CRISPR is the acronym for “clustered regularly interspaced short palindromic repeats” [3], a reference to the genomic locus of such repeating nucleotide sequences in bacterial and archaea [1]. Cas9 stands for “CRISPR-associated protein 9” [4], a CRISPR-associated endonuclease that is involved in the crCRNA-guided inactivation of foreign nucleic acids [3]. In 2012, Jinek et al. showed that it was possible to recruit Cas9 to specific genome loci through standard base pairing. In their study, Jenik et al. (2012) fused crRNA to the trans-activating crRNA (tracrRNA) of the Streptococcus pyogenes to generate a single guide RNA (sgRNA). The sgRNA was able to recruit Cas9 at specific genome loci and thus facilitating cleavage of sequences in these loci to achieve a desired effect [3]. This technology has been adopted in the development of therapeutic proteins that help in genome editing to inactivate mutated sequences that are linked to genetic diseases. This paper reviews such use of CRISPR-Cas9 to develop therapeutic proteins for various illnesses. 2. APPLICATIONS OF CRISPR-CAS9 FOR THERAPEUTIC PURPOSES Most of the studies published on use of CRISPR-Cas9 for therapeutic purposes relate to laboratory experiments with prokaryotes, eukaryotic cells (including humans), and experimental animals. However, the CRISPR-Cas9 technology is yet to be adopted in clinical use as evident from the lack of any clinical trials using the technology (based on search of trials for CRISPR-Cas9, CRISPR, Cas9 at www. clinicaltrials.gov/). Such failure to use the technology in clinical trials may be explained by the ethical issues accompanying genome editing in humans and uncertainty on the side effects of offtarget genome changes that may arise with use of genome-editing tools [5]. Specifically, editing of germ-line cells poses a Asian Journal of Pharmacy, Nursing and Medical Sciences (ISSN: 2321 – 3639) Volume 9 – Issue 1, February 2021 Asian Online Journals (www.ajouronline.com) 17 significant ethical dilemma since any genetic errors introduced in such cells can be passed on to subsequent generations and thus possibly leading to high prevalence of unforeseen genetic illnesses in the population [5]. Such concerns have limited the adoption of genome editing therapy in humans. For example, the first gene-editing case in people was done only recently, in 2014, using Zinc finger nucleases (SFNs) that target specific DNA sequences, to treat people with HIV [6]. Even then, such treatment has mainly entailed ex-vivo strategies, where gene edited cells are introduced into the patients’ bodies rather than in-vivo strategies, where the gene-editing tools are administered themselves instead of the altered cells using viral or non-viral vectors [4]. For CRISPR/Cas9 system, the most recent advance in their use in humanassociated cells has been the February 2016 approval for U.K. scientists to use the technology to edit human embryos for a duration of seven days, after which the embryos are to be destroyed [7]. The subsequent review of the therapeutic use of CRISPR-Cas9 system is thus based on laboratory data instead of human clinical trials. 3. USE OF CRISPR/CAS9 FOR ZYGOTE EDITING Use of CRISPR/Cas9 system to edit zygotes has been successfully tried in mice studies. In this approach, components of the CRISPR/Cas9 tool (e.g. Cas9 messenger RNA, sgRNA, and Homology Directed Repair (HDR) template; a strategy that seeks to correct errors in double-stranded DNA, are injected into the zygote or into an early-stage embryo [4]. In one such study, Wang et al. (2013) used the CRISPR/Cas9 tool to introduce mutations in mice through the co-injection of Cas9 mRNA and sgRNAs into mouse embryonic stem cells. Using this strategy, Wang et al. (2013) were able to target Tet1 and Tet2 genes leading to the production of mice biallelic mutations at an efficiency rate of 80% [8]. Further, when the researchers integrated mutant oligonucleotides into the sgRNAs that were co-injected with Cas9 mRNAs, they were able to induce point mutations in the two target genes in the resultant mice. This study demonstrated that the CRISPR/Cas9 editing tool could be used to generate mutations, which may be useful in evaluation of genes that are functionally redundant or in the evaluation of genes that have epistatic interactions. However, the study does not show whether CRISPR/Cas9 could be used to correct existing genetic disorders, a subject that was addressed in a different study reviewed subsequently [8]. In a different study, Wu et al. (2013) showed that the CRISPR/Cas9 editing tool could be used to correct mutations that led to cataracts in mice. In the study, Wu et al. (2013) co-injected zygotes of such mice with Cas9 mRNA and sgRNAs that targeted the Crygc gene. The study showed that the Cas9 system was able to repair the mutation in the Crygc gene based on HDR template oligonucleotides supplied by the researchers exogenously or based on endogenous wild-type-allele template [9]. The correction was noted to occur efficiently with limited off-target modifications, with the resultant mice retaining their fertility and successfully passing on the corrected allele to their offspring [9]. The results of this study thus showed that CRISPR/Cas9 was not only useful in generating mutations but also in correcting disease-causing mutations. Before the U.K. approval of a CRISPR/Cas9-based study using human embryo cells, an earlier study that had employed the technology on human embryo cells had resulted into divisions among scientists over the ethics of using such technology in embryo cells [5, 10]. In this earlier study, Liang et al. (2015) had used CRISPR/Cas9 to cleave endogenous β-globin gene (HBB, a gene implicated in causing β-thalassemia, a blood disorder) using Cas9 mRNA and sgRNA transfected into human triponuclear (3PN) zygotes. Nevertheless, the study found that CRISPR/Cas9 tool also resulted in high off-target effects (cleavage at non-targeted loci) and could introduce unwanted mutations due to the competition between exogenously supplied oligonucleotide template and the endogenous delta-globin gene [11]. Such results indicated that CRISPR/Cas9 may have low fidelity and specificity, a finding that led the researchers to recommend the non-use of CRISPR/Cas9 for clinical applications [11]. In summary, therefore, while the efficiency of CRISPR/Cas9 in mice embryos has been shown to be high with low off target effects, more research on the off target effects noted in human cells is needed before the technology can be reliably used in clinical applications targeting human embryo cells. Further, due to the possibility of use of CRISPR/Cas9 for non-medical purposes where embryos are involved such as altering traits of resultant children [4], use of CRISPR/Cas9 technology to edit human zygotes poses a grave ethical dilemma. 4. IN-VIVO EDITING OF SOMATIC CELLS USING CRISPR/CAS9 TECHNOLOGY While the editing of germ-line cells poses significant issues due to the possibility of transmitting any generated mutations to the progeny [5], editing of somatic cells poses a significantly lesser issue since any mutations arising from the process are not transmitted to the offspring. Such lower risk may explain the performance of in-vivo CRISPR/Cas9 tests targeting somatic cells, albeit in experimental animals. In one such study, Yin et al. (2014) showed that it was possible to use CRISP/Cas9 mechanism to correct for hereditary tyrosinemia typ

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