ADVANCE BIOTEC RESEARCH IN PAKISTAN DEFENSE LABS: GENE EDITING for BETTER & SMART LIVING, SECURITY,SAFE DELIVERY,EHICAL CONCERNS & MILITARY USES?

Worthy audience Genome editing, also called gene editing, is an area of research seeking to modify genes of living organisms to improve our understanding of gene function and develop ways to use it to treat genetic or acquired diseases. Genome editing can be used to correct, introduce or delete almost any DNA sequence in many different types of cells and organisms. Simply speaking, gene editing tools involve programmed insertion, deletion, or replacement of a specific segment of in the genome of a living cell. Potential targets of gene editing include repair of mutated gene, replacement of missing gene, interference with gene expression, or overexpression of a normal gene.

1   .Genome-wide editing is not a new field, and in fact, research in this field has been active since the 1970s.The real history of this technology started with pioneers in genome engineering. The first important step in gene editing was achieved when researchers demonstrated that when a segment of DNA including homologous arms at both ends is introduced into the cell, it can be integrated into the host genome through homologous recombination (HR) and can dictate wanted changes in the cell. Genome editing builds on an earlier discovery that a broken section of DNA in a gene triggers a cell’s repair mechanism to stitch together the break. Genome editing allows researchers to mimic this natural process of DNA repair. For the last 35 years Pakistan Armed Forces and Pak Institute of Health have made significant leap along with foreign scientist in promoting experiments on upgrading human life &elimination of lethality of the Battlefield ……rest is highly confidential. We must say well done Pak Army Institute of Advance Medical Research & NIH Pakistan.

 2 .Employing HR alone in genetic modification posed many problems and limitations including inefficient integration of external DNA and random incorporation in undesired genomic location. Consequently, the number of cells with modified genome was low and uneasy to locate among millions of cells. Evidently, it was necessary to develop a procedure by which scientists can promote output. Out of these limitations, a breakthrough came when it was figured out that, in eukaryotic cells, more efficient and accurate gene targeting mechanisms could be attained by the induction of a double stranded break (DSB) at a specified genomic. Scientists found that if an artificial DNA restriction enzyme is inserted into the cell, it cuts the DNA at specific recognition sites of double-stranded DNA (dsDNA) sequences. Thus, both the HR and non-homologous end joining (NHEJ) repair can be enhanced.

• Nuclease, any enzyme that cleaves nucleic acids. Nucleases, which belong to the class of enzymes called hydrolases, are usually specific in action, ribonucleases acting only upon ribonucleic acids (RNA) and deoxyribonucleases acting only upon deoxyribonucleic acids (DNA).
• The mode of action of what is known as site-directed nucleases is based on the site-specific cleavage of the DNA by means of nuclease and the triggering of the cell’s DNA repair mechanisms: HR and NHEJ.
• Various gene editing techniques have focused on the development and the use of different endonuclease-based mechanisms to create these breaks with high precision procedures (Fig.1) 

3 .Genome Editing Outcomes. Genome editing nucleases induce double-strand breaks (DSBs). The breaks are repaired through two ways: -

• Non-homologous end joining (NHEJ) in the absence of a donor template or via homologous recombination (HR) in the presence of a donor template. The NHEJ creates few base insertions or deletion, resulting in an indel, or in frame shift that causes gene disruption.
• In the HR pathway, a donor DNA (a plasmid or single-stranded oligonucleotide) can be integrated to the target site to modify the gene, introducing the nucleotides and leading to insertion of cDNA or frameshifts induction.
• One of the limitations in this procedure is that it has to be activated only in proliferating cells, adding that the level of activity depends on cell type and target gene locus.
• Tailoring of repair templates for correction or insertion steps will be affected by these differences. Several investigations have determined ideal homology-directed repair (HDR) donor configurations for specific applications in specific models systems.
• The differences in the activities of the DNA repair mechanisms will also influence the efficiency of causing indel mutations through NHEJ or the classical microhomology-mediated end joining (c-MMEJ) pathway, and even the survival of the targeted cells.
• The production of such repair in the cell is a sign of a characteristic that errors may occur during splicing the ends and cause the insertion or deletion of a short chainThe human genome developments paved the way to more extensive use of the reverse genetic analysis technique. Nowadays, two methods of gene editing exist: one is called “targeted gene replacement” to produce a local change in an existing gene sequence, usually without causing mutations. The other one involves more extensive changes in the natural genome of species in a subtler way.

4. Methods of Genes Editing . In the field of targeted nucleases and their potential application to model and non-model organisms, there are four major mechanisms of site-specific genome editing that have paved the way for new medical and agricultural breakthroughs. In particular, meganucleases (MegNs), zinc finger nucleases (ZFNs), transcription activator-like effector nuclease (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas-9).

• Advanced genome editing methods engineered from proteins include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases. An additional method is called clustered regularly interspaced short palindromic repeats, also known as CRISPR/Cas9.
• CRISPR/Cas9 is the most widely used genome editor and is a powerful tool for understanding gene function. Because CRISPR/Cas9 is an RNA-based system, it can be more efficiently and easily modified than the protein-based approaches and allows for targeting of multiple sites. CRISPR was discovered through NIH-funded basic research on how bacteria defend themselves from viruses. CRISPR/Cas9 works by cutting a DNA sequence at a specific genetic location and deleting or inserting DNA sequences, which can change a single base pair of DNA, large pieces of chromosomes, or regulation of gene expression levels. 
• Meganuclease (MegN) that generally cleaves its DNA substrate as a homodimer. 
• Zinc finger nuclease (ZFN) recognizes its target sites which is composed of two zinc finger monomers that flank a short spacer sequence recognized by the FokI cleavage domain.
• Transcription activator-like effector nuclease (TALEN) consists of two monomers; TALEN recognizes target sites which flank a fok1 nuclease domain to cut the DNA. 
• CRISPR/Cas9 system is made of a Cas9 protein with two nuclease domains: human umbilical vein endothelium cells (HuvC) split nuclease and the HNH, an endonuclease domain named for the characteristic histidine and asparagine residue, as well as a single guide RNA (sgRNA).

Fig. 2 Schematic diagram of the four endonucleases used in gene editing technologies.

5. Gene Delivery. From biotechnology’s point of view, the main obstacle that is facing molecular technology is to select the right method that is simple but effective to transfer the gene to the host cell. The components of gene editing have to be transferred to the cell/nucleus of interest using in vivo, ex vivo, or in vitro route. In this regard, several concerns must be considered including physical barriers (cell membranes, nuclear membranes) as well as digestion by proteases or nucleases of the host. Another important issue is the possible rejection by the immune system of the host if the components are delivered in vivo. In general, the gene delivery routes can be categorized in three classes of physical delivery, viral vectors, and non-viral agents. Although the direct delivery of construct plasmids may sound easy and more efficient and specific than the physical and the chemical methods, it proves to be an inappropriate choice because the successful gene delivery system requires the foreign genetic molecule to remain stable within the host cells. The other possible procedure is to use viruses. However, because plant cells have thick walls, the gene transfer systems for plants involve transient and stable transformation using protoplast-plasmid in vitro : agrobacterium-mediated transformation, gene gun and viral vectors (transient expression by protoplast transformation), and agro-infiltration. Viruses may present a suitable vehicle to transfer genome engineering components to all plant parts because they do not require transformation and/or tissue culture for delivering and mutated seeds could easily recovered. For many years, scientists employed different species of Agrobacterium to systematically infect a large number of plant species and generate transgenic plants. These bacterial species have small genome size and this facilitates cloning and agro infections, and the virus genome does not integrate into plant genomes .

• Of the challenges and approaches of delivering CRISPR, it was pointed out that although the present genome engineering is in favor of CRISPR tools, TALENs may still be of a primary choice in certain experimental species. For example, TALENs have been utilized in targeted genomic editing in Xenopus tropicalis by knocking-out Klf4 or thyroid hormone receptor α. In addition, TALENs have been utilized to modify genome of human stem cells . Also TALEN approach has been applied to create amniotic mesenchymal stem cells overexpressing anti-fibrotic interleukin-10 . Lately, a geminivirus genome has been prepared to deliver various nucleases platforms (including ZFN, TALENs, and the CRISPR/Cas system) and repair template for HR of DSBs .
• To deliver the carrying DNA sequence to target cells, non-viral techniques such as electroporation, lipofection, and microinjection can also be used. In addition, these techniques also reduce off-target cleavages problems. Gene transfer via microinjection is considered the gold standard procedure since its efficiency is approximately 100% . The advantage of this approach is its high efficacy and less constrains on the size of the delivery. A disadvantage is that it can be employed only in in vitro or ex vivo cargo. Recently, small RNAs, including small interfering RNA (siRNA) and microRNA (miRNA), have been widely adopted in research to replace laboratory animals and cell lines. Development of innovative nanoparticle-based transfer systems that deliver CRISPR/Cas9 constructs and maximize their effectiveness has been tested in the last few years .

6. Genome Editing in Research. Genome editing is widely used in studies in a variety of organisms. For example, CRISPR is used to make “knockout” models of disease in a wide range of animals, enabling researchers to study the underlying genetic causes. It also is being used to change genes in certain tissues or organs, facilitate the study of diseases by focusing on culprit genes, create cell models of disease such as in human pluripotent stem cells and inactivate viruses in pigs so that pig organs could potentially be used as a source of replacement organs for humans. It also is being explored to modify yeast cells to make biofuels and to improve strains of agricultural crops. Gene drive technology using CRISPR allows for the spread of engineered traits through populations of sexually reproducing organisms at a rate more rapid than what occurs in natural evolution. This research could be applied to alter mosquito populations to help interfere with the transmission of infectious diseases.

7. Genome Editing in the Clinic. NIH supports human gene therapy research, including genome editing approaches in somatic cells, for a wide array of diseases and conditions with grants, contracts and targeted efforts, such as the Somatic Cell Genome Editing Program. Somatic cells are any cells not involved in human reproduction. This means that changes in somatic cells are not inherited by subsequent generations. CRISPR and other gene editing methods, especially ZFNs, are speeding gene therapy approaches to treat many human conditions. In 2014, the first clinical application of genome editing involved the use of ZFNs to make human cells resistant to HIV-1 by disrupting a gene required for the virus to infect cells. In 2017, a clinical trial testing ZFNs to correct Hunter syndrome (MPS II) was launched. Caused by an enzyme deficiency, Hunter syndrome can cause abnormalities in the skeleton, heart and respiratory system. The clinical trial was the first genome editing approach administered directly to research participants. TALENs are being studied in T cell immunotherapy approaches to create “off-the-shelf” universal donor T cells that don’t have to be developed for each cancer patient. Genome editing approaches are also being pursued as part of NIH’s Cure Sickle Cell Initiative, and CRISPR is being used as a diagnostic tool to detect viruses such as Zika and dengue. In October 2019, NIH and the Bill and Melinda Gates Foundation announced a collaboration to support studies to advance the development of gene-based approaches to cure sickle cell disease and HIV. 

8. Ethical and Safety Concerns. As follows:-

• In 2017, the National Academies of Science, Engineering, and Medicine (NASEM) issued a report, Human Genome Editing: Science, Ethics, and Governance (2017),(link is external) which recommended that clinical trials using gene editing in embryos should be permitted only within a robust and effective regulatory framework and only when certain criteria are met.
• In November 2018, Chinese scientist He Jiankui announced that he had used the CRISPR/Cas9 technique to create the first genome edited babies, claiming to have disabled copies of a gene in the embryos to confer HIV resistance. This work raised serious questions of medical necessity, off-target effects and the potential for susceptibility to other infections. It led to nearly universal condemnation by the international scientific community, and the NIH Director issued a statement expressing serious concerns about the claim and urging continued international dialogue.
• On March 14, 2019, the NIH Director issued a statement supporting the call for an international moratorium on research establishing human pregnancies using germline-edited embryos. NIH (National Institute of Healh USA) continues to be involved in ongoing efforts by the World Health Organization (link is external) and sponsored a study of the NASEM/Royal Society of the United Kingdom(link is external) to examine the issues associated with human gene editing and develop global governance and oversight approaches. As discussed in the Director’s blog, the international commission concluded in the report, Heritable Human Genome Editing(link is external) (link is external) released
• In September 2020, that clinical use of heritable human gene editing should not be considered until it has been established that it is possible to make precise genomic changes efficiently and reliably without undesired changes in human embryos. If permitted, use should be limited to serious monogenic diseases. The report also included recommendations for scientific governance and oversight including the establishment of an international scientific advisory panel.
• These concerns are well addressed in current oversight structures. For instance, in the United States, well-established oversight frameworks and the U.S.
• Food and Drug Administration’s (FDA) regulatory authority over human gene therapy trials would apply to clinical research involving genome editing of somatic cells in humans. Additionally, biosafety guidance for the conduct of genome editing research is provided by the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. 
• The potential effects of gene drives on individual species and the environment have also raised biosafety, biosecurity and ethical concerns. A NASEM report, Gene Drives on the Horizon: Advancing Science, Navigating Uncertainty, and Aligning Research with Public Values (2016)(link is external), which was partially funded by NIH, explored the responsible conduct of gene drive research.
• In a 2016 statement by the NIH Director, NIH supported the report’s recommendations to continue basic and applied research on gene drives, hold off on current release of gene drive-modified organisms into the environment, have funders coordinate to encourage responsible development of gene drive technology and prioritize stakeholder engagement in decision-making. 

                          Images

In the gene-editing tool CRISPR, a small strand of RNA identifies a specific chunk of DNA. Then the enzyme Cas9 (green) swoops in and cuts the double-stranded DNA (blue/purple) in two places, removing the specific chunk.

CRISPR-Cas9 is a customizable tool that lets scientists cut and insert small pieces of DNA at precise areas along a DNA strand. The tool is composed of two basic parts: the Cas9 protein, which acts like the wrench, and the specific RNA guides, CRISPRs, which act as the set of different socket heads. These guides direct the Cas9 protein to the correct gene, or area on the DNA strand, that controls a particular trait. This lets scientists study our genes in a specific, targeted way and in real-time.

The CRISPR system has two components joined together: a finely tuned targeting device (a small strand of RNA programmed to look for a specific DNA sequence) and a strong cutting device (an enzyme called Cas9 that can cut through a double strand of DNA).

Once inside a cell, the CRISPR system locates the DNA it is programmed to find. The CRISPR seeking device recognizes and binds to the target DNA (circled, black).The Cas9 enzyme cuts both strands of the DNA.

Researchers can insert into the cell new sections of DNA. The cell automatically incorporates the new DNA into the gap when it repairs the broken DNA.

CRISPR has many possible uses, including insert a new gene so the organism produces useful medicines; help treat genetic diseases; create tailor-made organisms to study human diseases; and help produce replacements for damaged or diseased tissues and organs.

9. Base Editing & Prime Editing. Some of the most recently developed CRISPR methods are Base editing and Prime editing. These technologies work on the same principle, however at a more precise scale, inducing single nucleotide substitutions. Importantly, base editing and prime editing do not induce DSBs in the target DNA.

• Base Editing Involves. Either a catalytically dead Cas9 (dCas9) or a nickase Cas9 (nCas9). dCas9 is incapable of cutting DNA, while nCas9 produces ‘nicks’, or single-stranded breaks (SSBs) in the DNA. By fusing either dCas9 or nCas9 to a DNA modifying enzyme, researchers can alter specific nucleotides. One of the limitations of base editing is that they cannot be used to alter every possible nucleotide, and this is one of the factors that led to the development of prime editing.
• Prime Editing Involves. Fusing nCas9 to an engineered reverse transcriptase and a prime editing guide RNA (pegRNA). The pegRNA contains two sections: one that guides to the region of interest, and another that contains the desired substitution/s for repair after the single-stranded cut has been generated. After one strand has been altered by the prime editor, the complementary strand can also be corrected - an additional gRNA and nCas9 will create a nick in the strand and it will be repaired using the previously edited strand as a template. Prime editing is predicted to be capable of treating 89% of genetic mutations in humans.        

10. Important Applications of Crispr. In just a few short years, CRISPR has had a massive impact on scientific research, contributing to breakthroughs in medicine and biotechnology. Let’s take a closer look at some of the key applications of this technology.

·        Cell And Gene Therapies. CRISPR is poised to revolutionize medicine, with the potential to cure a range of genetic diseases, including neurodegenerative disease, blood disorders, cancer, and ocular disorders. As we mentioned earlier, the first trial of a CRISPR cell therapy was performed in 2019, treating patients with sickle cell disease. The treatment restored fetal hemoglobin, eliminating the need for a functional copy of adult hemoglobin.

·        In 2021, a significant CRISPR trial for transthyretin amyloidosis, a neurodegenerative disease, showed very promising results. It is also revolutionizing pediatric cancer treatment, described in this podcast interview with Shondra Miller from St Jude’s.CRISPR can also be used to generate chimeric antigen receptor (CAR) T cells, a form of immunotherapy used to treat cancer. The T cells are extracted from patients and engineered to express chimeric antigen receptors before being re-injected into the body.

·        The receptors allow the T cells to more efficiently target and destroy the specific type of cancer the patient suffers from. While we are still in the early years of clinical trials, this technology could be used to treat thousands of genetic conditions in the future, including breast and ovarian cancer linked to BRCA mutations, Huntington’s disease, Tay-Sachs, beta-thalassemia, cystic fibrosis, and early-onset Alzheimer’s. For all the latest medical developments and clinical trials using this technology to cure a range of human diseases, you can take a look at the CRISPR Medicine News website.

·              Diagnostics. During the COVID 19 pandemic, CRISPR was used as both a potential therapeutics tool and as a diagnostic tool for the coronavirus. The SHERLOCK™ CRISPR SARS-CoV-2 test kit was granted Emergency Use Authorization from the federal authorities to be used in laboratory settings. Like SHERLOCK and STOPCovid, DETECTR utilizes Cas9’s search function to detect genetic material from the virus, employing naturally occurring Cas nucleases, like Cas12 and Cas13. For more information on DETECTR .Similar diagnostics utilizing the search function of Cas9 have also been engineered to identify other diseases, both infectious and genetic. Early in 2021, Dr. Kiana Aran of Cardea Bio published a study which combined three different Nobel Prize-winning technologies graphene, transistors, and CRISPR - into a tiny chip that can detect pathogenic single nucleotide polymorphisms (SNPs). Since 50% of disease-causing mutations in humans are SNPs, this is a significant breakthrough in medical diagnostics.

·              Agriculture. Gene editing technology has huge potential in agriculture, and experts suggest that CRISPR-modified foods will be available within 5-10 years. This is primarily because it can be used to create crops that are disease-resistant and drought-resistant. For example, scientists from the University of Berkeley and Innovative Genomics Institute have partnered with Mars, Inc. to create disease-resistant cacao plants. It can also be used to prolong the shelf-life of other perishable foods, reducing food waste and allowing access to healthy foods at relatively low cost. For more information on these applications, you can read our overview of CRISPR’s use in agriculture.

·       Bioenergy. As one of the leading alternatives to fossil fuels, bioenergy has been under the spotlight for a while now. However, there are several hurdles to producing biofuels at scale. By using CRISPR, scientists have recently been able to make some significant advances in this area.For example, KO of multiple transcription factors that control production of lipids in algae has led to a huge increase in lipid production for generating biodiesel. Similarly, gene editing can improve the tolerance of yeast to harsh conditions during the production of biofuels. It has also increased editing efficiencies in bacterial species that are used to produce ethanol. For more details, you can check out this blog on how CRISPR is helping the biofuel industry.

11.             Applications Of Gene Technology. Genome/gene engineering technology is relatively applicable and has potential to effectively and rapidly revolutionize genome surgery and will soon transform agriculture, nutrition, and medicine. Some of the most important applications are briefly described below.

·  Plant-Based Genome Editing. One of the major goals for utilizing genome editing tools in plants is to generate improved crop varieties with higher yields and clear-cut addition of valuable traits such as high nutritional value, extended shelf life, stress tolerance, disease and pest resistance, or removal of undesirable traits .

·        Animal-Based Genome Editing. Recent genome editing techniques has been extensively applied in many organisms, such as bacteria, yeast, and mouse. Genetic manipulation tools cover a wide range of fields, including the generation of transgenic animals using embryonic stem cells (ESC), functional analysis of genes, model development for diseases, or drug development. Since the first permission to use CRISPR/Cas9 in human embryos and in vivo genome editing via homology-independent targeted integration (HITI), an increasing number of studies have identified striking differences between mouse and human pre-implantation development and pluripotency, highlighting the need for focused studies in human embryos.

·        At the beginning of 2019”, The “He Jiankui experiments which claimed to have created the world’s first genetically edited babies, is simply the most recent example. He Jiankui said he edited the babies’ genes at conception by selecting CRISPR/cas9 to edit the chemokine receptor type 5 (CCR5) gene in cd4+ cells in hopes of making children resistant to the AIDS virus, as their father was HIV-positive.

·        Researchers said He’s actions exposed the twins to unknown health risks, possibly including a higher susceptibility to viral illnesses. For more information on the scientific reactions around the world, the reader may find helpful several excellent sources of information.

12.  Gene Therapy. The original principles of gene therapy arose during the 1960s and early 1970s when restriction enzymes were utilized to manipulate DNA .Since then, researchers have done great efforts to treat genetic diseases but treatment for multiple mutations is difficult. Different clinical therapy applications have been attempted to overcome these problems. Much of the interest in CRISPR and other gene editing methods revolves around their potential to cure human diseases.

·        It is hoped that eradication of human diseases is not too far to achieve via the CRISPR system because it was employed in other fields of biological sciences such as genetic improvement and gene therapy. It is important to mention that the therapeutic efficiency of gene editing depends on several factors, such as editing efficacy, which varies widely depending on the cell type, senescence status, and cell cycle status of the target. Other factors that also influence therapeutic effectiveness include cell aptitude, which refers to the feasibility of accomplishing a therapeutic modification threshold, and the efficient transfer of programmable nuclease system to the target tissue, which is only considered to be effective if the engineered nuclease system reaches safely and efficiently to the nucleus of the target cell. Finally, the precision of the editing procedure is another important aspect, which refers to only editing the target DNA without affecting any other genes .

·        Preliminary experiments, the knocking-in procedure was used to reach this goal. There are examples of gene editing techniques applied in different genetic diseases in cell lines, disease models, and human.

·        These encouraging results suggest the therapeutic capability of these gene editing strategies to treat human genetic diseases including Duchenne muscular dystrophy, cystic fibrosis, sickle cell anemia, and Down syndrome.

·        In addition, this technology has been employed in curing Fanconi anemia by correcting point mutation in patient-derived fibroblasts, as well as in hemophilia for the restoration of factor VIII deficiency in mice. The CRISPR tools have also demonstrated promising results in diagnosis and curing fatal diseases such as AIDS and cancer

·        The genome editing tools have enabled scientists to utilize genetically programmed animals to understand the cause of various diseases and to understand molecular mechanisms that can be explored for better therapeutic strategies (Fig. 7). Genome editing gives the basis of the treatment of many kinds of diseases.

·        Other Applications. The applications mentioned above were more about knock out or modification of genes Gapinske et al. However due to inactivate nuclease activity nature of the dCas9, CRISPR can be used in other applications as well. By selecting the target sequence, gene expression can be controlled by inhibiting the transcription rate of RNA polymerase II (polII) or inhibiting the transcription factor binding .Additionally, combining gene expression inhibitors such as Krüppel-associated box with the inactivated Cas9 has led to generate a special kind of gene inhibitors, which are called CRISPR interference (CRISPRi), and downregulate gene expression . It is also possible to control gene expression by fusing transcription-activating molecule, the transcription-repressing molecule, or the genome-modifying molecule to dCas9 .

Top: In the ex vivo editing therapy, cells are removed from a patient to be treated, corrected by gene editing and then re-engrafted back to the patient. To achieve therapeutic success, the target cells must be capable of surviving in vitro and autologous transplantation of the corrected cells.

Below: In the in vivo editing therapy, designed nucleases are administered using viral or non-viral techniques and directly injected locally to the affected tissue, such as the eye, brain, or muscle. (Adapted from)

12.     Conclusions. Genome editing is a fast-growing field. Editing nucleases have revolutionized genomic engineering, allowing easy editing of the mammalian genome. Much progress has been accomplished in the improvement of gene editing technologies since their discovery. Of the four major nucleases used to cut and edit the genome, each has its own advantages and disadvantages, and the choice of which gene editing method depends on the specific situation. The current genome editing techniques are still buckling up with problems, and it is difficult to perform genome editing in cells with low transfection efficiency or in some cultured cells such as primary cultured cells. Genotoxicity is an inherent problem of enzymes that act on nucleic acids, though one can expect that highly specific endonucleases would reduce or abolish this issue. Exceptional efforts are needed in future to complement and offer something novel approaches in addition to the already existing ones. It is anticipated that research in gene editing is going to continue and tremendously advance. With the development of next-generation sequencing technology, new extremely important clinical applications, such as manufacturing engineered medical products, eradication of human genetic diseases, treatment of AIDS and cancers, as well as improvement of crop and food, will be introduced. Combination of genomic modifications induced by targeted nucleases to their own self-degradation, self-inactivating vectors may help overcoming confronting limitations discussed above to improve the specificity of genome editing, especially because the frequency of off-target modifications. Our understanding of off-target effects remains poor. This is a vital area for continued study if CRISPR/Cas9 is to realize its promise.

13.   Regarding gene cargo delivery systems, this remains the greatest obstacle for CRISPR/Cas9 use, and an all-purpose delivery method has yet to emerge. The union between genome engineering and regenerative medicine is still in its infancy; realizing the full potential of these technologies in reprograming the fate of stem/progenitor cells requires that their functional landscape be fully explored in these genetic backgrounds. Humankind can only wait to see what the potential of these technologies will be.One major question is whether or not the body’s immune response will accept or reject the foreign genetic elements within the cells. Another important concern is that along with the revolutionary advances of this biotechnology and related sciences, bioethical concerns and legal problems related to this issue are still increasing in view of the possibility of human genetic manipulation and the unsafety of procedures involved. The enforcement of technical and ethical guidelines, and legislations should be considered and need serious attention as soon as possible.

Abbreviations

Cas-9 : CRISPR-associated protein 9

CRISPR: Clustered regularly interspaced short palindromic repeats

crRNA: CRISPR RNA

DSB: Double-stranded break

ESC: Embryonic stem cells

HDR: Homology-directed repair

HITI: Homology-independent targeted integration

HR: Homologous recombination

HuvC: Human umbilical vein endothelium cells

I-Sce I: Intron-encoded endonuclease

MegNs: Meganucleases

MMEJ: Microhomology-mediated end joining

NHEJ: Non-homologous end joining

PACE: Phage-assisted continuous evolution

PAMs: Protospacer adjacent motifs

RGENs: RNA-guided endonucleases

RVD: Repeat variable di-residues

sgRNA: Single guide RNA

SpyCas9: Streptococcus pyogenes Cas9

SSB: Single-strand break

TALENs: Transcription activator-like effector nuclease

ZFNs: Zinc finger nucleases