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Received: 18 May 2018 | Accepted: 4 September 2018 DOI: 10.1002/jcp.27476 R EV I EW ART I C L E CRISPR–Cas9 in genome editing: Its function and medical applications Saedeh Khadempar1 | Shokoufeh Familghadakchi2 | Roozbeh Akbari Motlagh3 | Najmeh Farahani4 | Maryam Dashtiahangar5 | Hamzeh Rezaei6 | Seyed Mohammad Gheibi Hayat7 1Departemant of Medical Genetics, Shahid Sadoughi University of Medical Science, Yazd, Iran 2Department of Clinical Biochemistry, School of Paramedicine, Hamadan University of Medical Sciences, Hamadan, Iran 3Department of Biochemistry and Molecular Biology, Faculty of Veterinary, Shahid Chamran University of Ahvaz, Ahvaz, Iran 4Department of Genetics and Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran 5Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran 6Department of Clinical Biochemistry, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran 7Department of Medical Biotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Correspondence Seyed Mohammad Gheibi Hayat, Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad 91779‐48564, Iran. Email: Gheibi65@yahoo.com Abstract The targeted genome modification using RNA‐guided nucleases is associated with several advantages such as a rapid, easy, and efficient method that not only provides the manipulation and alteration of genes and functional studies for researchers, but also increases their awareness of the molecular basis of the disease and development of new and targeted therapeutic approaches. Different techniques have been emerged so far as the molecular scissors mediating targeted genome editing including zinc finger nuclease, transcription activator‐like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR‐associated protein 9 (Cas9). CRISPR–Cas9 is a bacterial immune system against viruses in which the single‐strand RNA‐guided Cas9 nuclease is linked to the targeted complementary sequences to apply changes. The advances made in the transfer, modification, and emergence of specific solutions have led to the creation of different classes of CRISPR–Cas9. Since this robust tool is capable of direct correction of disease‐causing mutations, its ability to treat genetic disorders has attracted the tremendous attention of researchers. Considering the reported cases of nonspecific targeting of Cas9 proteins, many studies focused on enhancing the Cas9 features. In this regard, significant advances have been made in choosing guide RNA, new enzymes and methods for identifying misplaced targeting. Here, we highlighted the history and various direct aspects of CRISPR–Cas9, such as precision in genomic targeting, system transfer and its control over correction events with its applications in future biological studies, and modern treatment of diseases. K E YWORD S CRISPR–Cas9, genome editing, new treatments, specific targeting 1 | INTRODUCTION In recent years, the introduction of programmable nucleases has greatly increased the efficiency of targeted genome editing. The programmable nucleases can be classified into four main classes. Before the development of technology, researchers used different CRISPR tools capable of breaking double‐stranded DNA to generate variations in the genome. For example, DNA‐binding nucleases including meganuclease (MN; Xu, Liu, & Pardinas, 2015), zinc finger nuclease (ZFN; Jo, Kim, & Ramakrishna, 2015), transcription activator‐like effector nucleases (TALEN; Scharenberg, Duchateau, & Smith, 2013), J Cell Physiol. 2019;234:5751–5761. wileyonlinelibrary.com/journal/jcp © 2018 Wiley Periodicals, Inc. | 5751 clustered regularly interspaced short palindromic repeats (CRISPR)– CRISPR‐associated protein 9 (Cas9; Go & Stottmann, 2016) are the four most recently discovered type types. The function of all these tools is to identify and link specifically to the target DNA and then create a double‐strand break (DSB). In the first three classes, detecting DNA‐binding domain is derived from protein. As a result, the use of these systems due to the need to design and engineer the protein in any experiment is very difficult, time‐consuming, and costly. The CRISPR–Cas system is able to detect target DNA‐binding domain derived from RNA, so the system is very rapid, simple, and inexpensive. The CRISPR–Cas is a bacterial adaptive immune system which first discovered in 1987 (Lander, 2016). Each of the above‐mentioned nucleases has been subject to limitations. The MNs are similar to restriction enzymes and are designed to be capable of targeting sequences of 14–40 bp. The MNs have been used only for a short time due to deficiencies such as inadequate specificity in detecting the target DNA and the need for their restriction sites to integrate with the target sequence. ZFNs and TALENs have relatively similar functions, although they differ in DNA‐binding sites, and include trinucleotide and mononucleotide patterns. These enzymes have separate DNA‐binding sites and a nonspecific restriction site called FokI endonuclease. These char- acteristics, however, have led to the superiority of these two enzymes compared with MNs (Silva et al., 2011). The biggest challenge for designing and manufacturing of ZFNs is the feature of specific binding to the target. In the case of TALENs, in spite of having a binding property, each of the TALE domains and each of the target sequence bases require sophisticated molecular cloning techniques to design the determined and conserved TALE compo- nents. Finally, the advent of powerful CRISPR introduced far more cost‐effective and easier methods for generating changes in the genome and protein engineering compared with the earlier methods (Figure 1; Joung & Sander, 2013). Accurate and targeted editing capability at any spot in the genome of beings for many years has been the aspiration of scientists, and today scientists have come closer to this goal with the discovery of CRISPR (Hsu, Lander, & Zhang, 2014). Researchers are now able to knockout and knockin any gene in the genome. The genome editing based on the CRISPR–Cas9 system is an efficient and high‐potential tool that can be replaced by old methods such as ZFN and TALEN (Sander & Joung, 2014). 2 | THE Cas9 AS AN IMMUNE SYSTEM IN BACTERIA AND ARCHAEA Ishino Y et al. (1987) identified CRISPR in Escherichia coli. They found that these zones have a specific barcode called spacers with matches originate from viral or plasmid genomes. The hypothesis of the involvement of these repeats in the adaptive defense of prokaryotes was confirmed in 2007. In other words, the spacer sequence is formed in the bacterial genome during exposure to phages, and the level of bacterial sensitivity to be infections by phages depends on the contents of the spacer sequence. Subsequent studies showed that the CRISPR works as a complex with Cas9 endonuclease, so that the Cas9 protein‐encoding gene is located in the vicinity of the CRISPR locus. The Cas9 creates a gap in the target DNA or RNA sequences. The bacteria and archaea via CRISPR–Cas9 protect their genome against the attack of the phage nucleic acid and the integrating plasmids. In fact, CRISPR–Cas9 comes with the help of the immune system and targets a number of invading nucleic acids and proteins, including DNA and RNA, by coordinated activity (Hale et al., 2009). The invasive foreign DNA is broken down by the Cas nucleases, and then part of it is placed in the CRISPR site between two repeated sequences, in which case it is referred to as a spacer. The spacersequences are used as templates for the production of short CRISPR RNAs (crRNAs) and form a complex with the transactivating crRNA (tracrRNA) molecule (Brouns et al., 2008; Jinek et al., 2012). These two sequences together, as a guide sequence, direct the Cas9 protein towards the invasive DNA. Upon the Cas9 protein binds to the invasive DNA, this protein cleaves the foreign DNA strand complementary to the crRNA sequence and its opposite sequence through the nuclease domains of NHN and RuvC1‐Like, respectively (F. Jiang et al., 2016). The CRISPR–Cas9 systems can be classified based on the performance of their subunits into two main classes. The first class of the CRISPR–Cas9 systems consisting of multisubunit effector RNA complexes (types I, III, and IV), and the second CRISPR–Cas9 systems are composed of single‐subunit RNA effector consisting of types II and V (Figure 2; Barrangou et al., 2007). 3 | A TOOL FOR GENOME EDITING The modern targeted genome editing system using the CRISPR–Cas9 technology has two components: an endonuclease and guide RNA with short sequence (Jinek et al., 2012). The targeted endonuclease is a bacterial Cas9 enzyme derived from Streptococcus pyogenes. The Cas9 nuclease has two DNA cleavage domains: HNH nuclease domain and RuvC1‐like nuclease domain (F. Jiang et al., 2016), leading to blunt DNA DSB (Figure 3). The guide RNA (gRNA) in this system refers to the engineered chimeric single‐strand RNA, which has both tracrRNA and bacterial crRNA roles. The researcher designs 20‐bp 5′‐terminal nucleotides related to the gRNA (homing device) against the gene sequence desired for cleavage and editing. This 24‐nucleotide sequence guides the Cas9– gRNA complex to the targeted gene location just upstream of the protospacer adjacent motif (PAM) site via RNA–DNA binding. The PAM sequence differs between the different bacterial strains and the various CRISPR–Cas proteins, and this sequence is 5′‐NGG for S. pyogenes. Therefore, the CRISPR–Cas system in S. pyogenes is directed to each DNA sequence with 5′‐N20‐NGG and accurately blunt DNA DSB (Garneau et al., 2010). The sequence detected by PAM occurring via Cas9 enzyme is different based on the bacterial strains producing the Cas9 nucleases. The Cas9 nuclease in S. pyogenes is a type II system and 5752 | KHADEMPAR ET AL. most commonly used for genome editing (Makarova et al., 2015). It should be noted that other commercial Cas9 enzymes detect other PAM sequences (Ran, Hsu, Wright et al., 2013; Figure 4). The DNA DSB repair occurs through two mechanisms which could be observed in all types of organisms, called nonhomologous end‐joining (NHEJ) and homology‐directed repair (HDR) pathways (Maruyama et al., 2015). The successful genome editing using CRISPR–Cas9 depends on the gRNA and the PAM sequences, and only the target sequence that immediately follows the PAM sequence is targeted for genome editing. 4 | REGULATION OF GENE EXPRESSION There are several ways to change the gene expression level, such as epigenetic changes, transcription factors, and others. One of the main challenges of driving these factors specifically toward the gene is to change the expression of a gene in the use of transcription factors. The CRISPR system, which could target exclusively a specific site in the genome, made it possible for researchers who direct the transcription factors specifically towards a gene to alter the expression of that gene. All of these methods are based on a general principle, which is the link between the gene expression modifying agent and the Cas9 passive protein. Because we tend to change only the gene expression in this way, so we make mutation in the Cas9 protein to deactivate the nuclease domains, which is called dCas9. As a result, the dead Cas9 (dCas9) is unable to cleave the DNA. 5 | DOWNREGULATION OF GENE EXPRESSION The first successful results of the gene expression downregulation using dCas9 were observed in E. coli. One of the easiest methods to F IGURE 1 Insertion of foreign genomic segments in repeated sequences (a,b). crRNA: clustered regularly interspaced short palindromic repeats RNA; tracrRNA: transactivating‐crRNA [Color figure can be viewed at wileyonlinelibrary.com] KHADEMPAR ET AL. | 5753 turn the gene off is to select the gRNA so that dCas9 is located on the downstream of the transcription start site, thus preventing the transcription elongation step by the RNA polymerase (Bikard et al., 2013; Larson et al., 2013). This kind of CRISPR system that causes the inhibition of gene expression is called CRISPR interference. Scientists were able to use dCas9 for reducing slightly the enhanced green fluorescent protein (egfp) gene expression in the HEK293T cell line. The use of dCas9 coupled with a gene expression inhibitor could increase the system efficiency to reduce gene expression. Therefore, they used the domains of transcriptional inhibitors such as the Krüptel‐associated box (or by binding four domains of mSin3 and forming SID4X) to reduce by more than 80% the gene expression of the transferrin receptor CD71, C‐X‐C chemokine receptor type 4 (CXCR4), and tumor protein 53 (TP53). The method of binding these transcriptional inhibitors is to link these agents to the protein C‐terminal using the Cas9 protein engineering (Gilbert et al., 2013). In another way to further reduce the gene expression, in addition to the protein C‐terminal, a transcriptional inhibitor was attached to the protein N‐terminal as well (Gilbert et al., 2014). 6 | UPREGULATION OF GENE EXPRESSION In a simple method to increase the gene expression in E. coli, the ω subunit of the RNA polymerase was linked to the dCas9 protein, allowing the assemblage of the holoenzyme in the target promoter (Bikard et al., 2013). The gene expression in mammalian cells can be increased using VP64‐activation agents (a complex resulting from the binding of four VP16 proteins) and other transcription activators. To further increase the expression level, the activator attaches to both N‐ and C‐terminals of protein (Farzadfard, Perli, & Lu, 2013; Gao et al., 2014). In a sophisticated but efficient way to far more gene expression, they attached 10 peptides as epitopes, and then removed the scFV domain of antibody specific for these epitopes and gave them VP64 or other activators. Therefore, the decapeptide complex was attached to the C‐terminal of the dCas9 protein, which is called SunTag array (Tanenbaum, Gilbert, Qi, Weissman, & Vale, 2014). In another approach to activate transcription, the gRNA is engineered instead of the dCas9 protein, and RNA molecules called aptamers binding to a protein instead of some of the loops. This approach uses MS2 aptamer, such proteins that are bound to this RNA are called MS2 coat proteins. In this method using engineering, the MS2 coat protein is linked to transcription activator or nonactivator (Konermann et al., 2015). F IGURE 2 Types and classes of CRISPR–Cas9 systems. CRISPR–Cas9: clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR‐ associated protein 9; crRNA: CRISPR RNA; PAM: protospacer adjacent motif [Color figure can be viewed at wileyonlinelibrary.com] F IGURE 3 Nuclease domains of Cas9. Cas9: clustered regularly interspaced short palindromic repeats‐associated protein 9; gRNA: guide RNA; PAM: protospacer adjacent motif [Color figure can be viewed at wileyonlinelibrary.com] 5754 | KHADEMPAR ET AL. 7 | APPROPRIATE DESIGN OF GRNA FOR THE CRISPR–Cas9 SYSTEM Recent advances in CRISPR–Cas9 in the in vitro, in vivo, and even in animal models, such as rat, zebrafish, and monkeys, have led to its widespread usage. It is noteworthy that, for other programmable nucleases including ZFN, TALEN,and MN, the nonspecific nuclease conduction to the target site on the genome is based on the DNA– protein interaction. In the CRISPR–Cas9 system, this happens based on the DNA binding to the target RNA; therefore, this system can create a massive off‐target cleavage sites in the genome (Zhang, Tee, Wang, Huang, & Yang, 2015). In addition to the large amounts of off‐target cleavage sites on the upstream sites of the PAM with ‐NGG sequences, the possibility of off‐ target cleavage sites has been observed on sites including the PAM secondary sequence (NAG) for SpCas9 (Hsu et al., 2013). Consequently, comprehensive studies have been done to design online databases to identify potentially off‐target cleavage sites based on the sequence similarity to the target site as well as mismatches along the crRNA (Graham & Root, 2015). The proper gRNA design for the CRISPR–Cas technique is one of the key steps to prevent off‐target cleavage sites. Factors such as the specific position of nucleotides along gRNA, surrounding site sequence, gRNA subsidence, guanine–cytosine (GC) content along gRNA, and the appropriate secondary structure for gRNA should be considered in design to achieve cleavage at the appropriate site (Doench et al., 2016). A study examined 218 different gRNAs at different sites of the genome and monitored their activity, and determined that 3′ and also 20 (nucleotide adjacent to PAM) positions were clearly associated with appropriate cleavage in 20‐nucleotide gRNA sequence (Doench et al., 2014). In addition, structural studies have shown that the 20‐nucleotide segment plays an important role in the formation of the F IGURE 4 Schema of CRISPR–Cas genome editing technology (a-g). CRISPR–Cas9: clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR‐associated protein 9; dCas9: dead Cas9; DSB: double‐strand break; GFP: green fluorescent protein; HDR: homology‐ directed repair; NHEJ: nonhomologous end‐joining; PAM: protospacer adjacent motif; sgRNA: single guide RNA; ssRNA: single‐stranded RNA [Color figure can be viewed at wileyonlinelibrary.com] KHADEMPAR ET AL. | 5755 primary loop R, a triple‐stranded structure including gRNA hybrid with target DNA strands when two DNA strands are broken up (Anders, Niewoehner, Duerst, & Jinek, 2014). Adenine in this site has also been reported to reduce cleavage rates by about 50%. In general, nucleotides in the seed region are important in the proper cleavage efficiency (Gagnon et al., 2014). Initially, it was believed that the nonseed sequence was not decisive for the proper cleavage, but recent studies showed that the presence of thymidine and guanine nucleotides at positions 2 and 3 would have a negative effect on the proper cleavage. It has also been indicated that the adenine at position 6 of this sequence plays an important role in the proper cleavage efficiency. Generally speaking, the nucleotides at 2 and 4–6 positions in gRNA are pivotal in binding to the target site, resulting in better recruitment of Cas9 protein towards RNA/DNA heteroduplex. In the statistical study of 218 gRNAs, as mentioned earlier, it was observed that low or high GC content had negative effects on proper cleavage and about 40–60% GC content was needed for proper cleavage (Liu et al., 2016). The genomic position of the gRNA binding to DNA also plays an important role in cleavage, and it has been observed that the RNA binding to the proximal promoter and the transcription onset are more efficient than in the intergenic regions, since chromatin is more available in these regions (Singh, Kuscu, Quinlan, Qi, & Adli, 2015). The formation of the secondary structure by gRNA, especially in the seed sequence, is important to recruit the Cas9 to the target site and also induces the active endonuclease structure of this enzyme (Jinek et al., 2014). One of the approaches proposed to reduce off‐target cleavage is the use of truncated gRNA (tru‐gRNA), which has about 17–18 bases. The tru‐gRNA produces less off‐target cleavage (about 500 times lower) compared with the common 20‐nucleotide gRNA (Fu, Sander, Reyon, Cascio, & Joung, 2014). This shortening should be made at the gRNA 5′‐terminal since shortening at the 3′‐terminal results in a decrease and even loss of gRNA activity (Fu et al., 2013). Reducing more than three nucleotides reduces or eliminates gRNA activity. It seems that the common 20‐nucleotide gRNA generates more energy for Cas9, while tru‐gRNA produces minimal energy for Cas9, thus preventing off‐target cleavage. The gRNA with additional guanine at the 5′‐terminal (5′‐GGX20 or GGGX 19‐5′) can be more specialized. Although there is still no precise mechanism for this issue, the change in the gRNA stability, its expression level or its secondary structure can be considered (Kim et al., 2015). Some other factors that increase the probability of a specific binding are the addition of the A‐U base pair to the gRNA scaffold (the tracrRNA sequence) that stabilizes the scaffold and the addition of the base pair to the hairpin loop in the spatial structure of the scaffolding that increases the gRNA binding rate to the target site and ultimately enhances the specific cleavage (Chen et al., 2013). Figure 3 summarizes the stages of gRNA design. 8 | MORE EFFICIENT AND MORE ACCURATE TYPES OF CRISPR SYSTEM Recent studies have shown that although every 20 nucleotides in the gRNA sequence are necessary for specific function and binding to the target, a few mismatches are tolerated in these locations, in turn causing the off‐target cleavage in the genome (Cong et al., 2013; W. Jiang, Bikard, Cox, Zhang, & Marraffini, 2013). The Cas9 nickase has been introduced to prevent the double‐strand cleavage in nonspecific site. As noted, the Cas9 protein has two cleavage domains, each one creating cleavage in one of the DNA strands, so deactivating each of these domains can provide an enzyme enabling single‐strand nick, which is called nickase. In this case, the double‐strand cleavage needs to be induced by two gRNAs, each linked to one of the strands, such that the 5′‐terminal of these gRNAs is in the same direction and the distance between these two 5′ offsets is 0–20 bp. In such a case, the cleavage is created with an additional 5′‐terminal in the target DNA. This system is known as double nicking. In this system, the nickase with D10A mutation in the RuvC domain is used normally (Ran, Hsu, Lin et al., 2013). The importance of using the nickase is that if any of the gRNAs were nonspecifically linked to the site and created a single‐strand nick, then the BEC (base excision repair) system would repair it, and only the double‐strand cleavage created as a result of binding two gRNAs in close proximity leads to the activation of NHEJ or HDR pathways. It should be noted that no effective method has yet been introduced to check the effects of a single‐strand nick. This system significantly reduces the off‐target cleavage through the performance of Cas9n is also slightly reduced compared to the wild type of the Cas9 protein (Mali et al., 2013). To improve the nickase system, the researchers introduced a more precise type of Cas9, in which both cleavage domains were deactivated. This type is called dCas9. Subsequently, the nonspecific Fok 1 cleavage domain binding to the dCas9 N‐terminal develops a system called RNA‐ guided Fok 1 nuclease (RFN). In this system, the dCas9 acts as a carrier and a director of Fok 1 to the cleavage position. The principle of the system is similar to double nicking, with the difference that the Fok 1 nuclease activity requires dimerization with its counterpart in the opposite strand; therefore, unlike the nickase system, the gRNA binding in RFN alone will not lead to a single‐strand nick. Likedouble nicking, the system also requires the design of two gRNAs, each one connected to one of the DNA strands. In this case, 5′‐terminal of these gRNAs should be toward each other and the effective distance between the 5′ gRNAs is bp (Tsai et al., 2014). In addition, a new system for simultaneous expression of two or more gRNAs has been used to optimize RFN as much as possible. In this system, the endoribonuclease Csy4 is expressed along with the dCas9–FokI complex in the cell. The cleavage site of this enzyme is also located on the upstream region of the gRNA‐encoding sequence. In this way, the gRNAs are expressed as a transcript under a single promoter, and then the Cys‐4 enzyme separates them by creating cleavage at their own site. In conventional gRNA expression systems, the guanine is necessary to be designed before 5′ region of the gRNA because this design is essential for the effective expression of RNA from the U6 promoter, whereas in the Cys‐4‐based expression model, 5′ region of the transcript contains the first detection sequence of Cys‐4. Therefore, the presence of guanine is not compulsory in the 5′ region of the gRNA. This issue is very important in the design of gRNA because it broadens the range of gRNAs appropriate for the 5756 | KHADEMPAR ET AL. target (Cheng et al., 2013; Guilinger, Thompson, & Liu, 2014). The simultaneous use of tru‐gRNA and RFN in cancer cell lines and stem cells provides a good cleavage at high levels (Wyvekens, Topkar, Khayter, Joung, & Tsai, 2015). One of the major limitations of this system is its large size that prevents it from being transmitted using viral vectors (Tsai & Joung, 2016). One of the important issues in the development of off‐target cleavage in the genome is the prolonged expression of the components in the CRISPR system, especially Cas9 in the cell, because this raises the possibility of inducing off‐target cleavage. The researchers assessed the crystalline structure of this protein and explored a position for splitting the protein into two fragments without affecting the cleavage domains. This new form, called Split Cas9, has the capability of reassembling after exposure to gRNA. However, it has been shown that the activity of this enzyme is incompatible with the tru‐gRNA system (Wright et al., 2015). Subsequently, to eliminate the dependence of the activity of the Split Cas9 system on gRNA, dimerizable domains inducible with photon or small molecules were used to induce dimerization of this Cas9 types. Zetsche et al. applied the rapamycin‐dimerizing domain. This domain makes it possible to assemble Cas9 in the presence of rapamycin (Zetsche, Volz, Zhang, 2015). In another study, Davis et al. inserted an intein inducible with 4‐hydroxyta- moxifen (4‐HT) in the appropriate site in the Cas9 structure. This intein only is excluded from the enzyme in the presence of 4‐HT. As a result, the Cas9 enzyme is activated in the presence of inducible compound and deactivated in the absence of the compound. This increases the specific system up to 25% in human cells (Davis, Pattanayak, Thompson, Zuris, & Liu, 2015). In addition, a photoactivatable Cas9 has also recently been reported. This type has been developed based on the adhesion of a photoinducible dimerizing domains to the Split Cas9 subunits. The obvious feature of this system is the reversibility that cannot be found in rapamycin‐ or intein‐inducible systems (Nihongaki, Kawano, Nakajima, & Sato, 2015). Replacing the amino acid in the wild Cas9 sequence can increase the binding power of this protein and thus cause intolerance to mismatch in the gRNA sequence. The eSpCas9 is a variant of SpCas9 in which three alanine amino acids are replaced (A/K1003A/ R1060A848K). The tails are linked to the off‐target strand. This replacement led to removal of off‐target cleavages in the genome and also increased cleavage in the specific site compared with the wild form (Slaymaker et al., 2016). In another study, the replacement of four amino acids in the nonspecific binding site of the target DNA strand with gRNA (N497A, R661A, Q695A, and Q926A) increased binding energy, and so reduced significantly the off‐target cleavage in the genome (Kleinstiver et al., 2015). Applying these two types of enzymes with the same gRNA and in the same cell line will be very significant to compare their function. The introduction of these two new types has been a great help for more precise genome engineering for the level of genetic disease treatment. The discovery and introduction of other proteins with endonu- clease activity used for genome engineering can provide more comprehensive insights for the effective application of this tool, including Cpf1 protein whose function in human genome engineering has recently been studied (Kim et al., 2016). The Cpf1 is another popular variant of the Class II CRISPR system, named as the type V. This endonuclease has structural and functional differences with the Cas9: 1. Its activity is dependent only on the presence of a crRNA and needs no tracrRNA, 2. Creation of a terminal cleavage that increases the insertion rate of a gene cassette to the genome, 3. The PAM position is 5′‐TTTN‐3′, which is rich in thymidine, 4. Cpf1 only contains a cleavage domain as similar to RuvC (Zetsche, Gootenberg et al., 2015). Recently, it has been observed that this protein (Cpf1) has both DNase and RNase properties, which cause the editing of the precursor RNA to direct towards the target protein (Fonfara, Richter, Bratovič, Le Rhun, & Charpentier, 2016). The crystalline structure of this protein has also been well studied in the target site, which can provide a comprehensive view of its functions (Kim et al., 2016; Zetsche, Gootenberg et al., 2015). 9 | INTRODUCTION TO CRISPR–Cas9 APPLICATIONS The CRISPR–Cas9 method is often used for the dual inactivation of the gene pair and detecting intergene correlation though this method can be applied to investigate the response of cells in exposure to drugs. The protein product of this correlation can be independently investigated by protein assay techniques. Finally, the repair of this correlation framework in eukaryotic cells can help understand the set of messenger pathways involved in cancer, and thus how the intergene correlation network affects tumorigenicity. In contrast, the sensitivity level of recent treatments can be improved by using lethal connections induced by new drugs. In this regard, the important role of gRNA in this collection should be given special attention and efforts should be made to improve this system by promoting its design. In this way, it can increase the rate of specific editions, reduce the number of false positive targets, and design the gRNA that targets the functional protein domains. Since the difference in the Cas9 expression between cells in a line and other different lines affects the effectiveness of correlation and impair- ment, several studies, especially on proteomics, are needed to provide a more comprehensive map of this correlation framework. It should be emphasized that this experimental work framework alone is unusable to cancer cells, but it can be applied for studying complex biosystems and diseases in a variety of eukaryotic cells with the ability of lentiviral transduction and cultivation in the media (Canver et al., 2017). Clearly, all cancers are the result of numerous and diverse mutations that lead to cell overproliferation and the appearance of KHADEMPAR ET AL. | 5757 malignant phenotypes. The event bed and the impaired domain of these mutations can be classified into four distinct categories: oncogenes, tumor suppressors, epigenetic agents, and chemotherapy resistance‐inducinggenes (Young et al., 2017). The above characteristic has the ability to correct these mutations and to treat partly their resulting cancer. Since the oncogenetic changes in a number of cancers lead to increased cell proliferation and malignancy, it is possible to target oncogenes, such as the receptor tyrosine kinase ErbB2, directly using the CRISPR– Cas9 method. In a point of view, the CRISPR–Cas9 method can create carcinogenic mutations in human cell lines and animal models. In this regard, the cell lines have been developed for lung cancers, acute myeloid leukemia, liver cancer, and pancreatic cancer (Xue et al., 2014). The CRISPR–Cas9 systems can also be used to systematically examine the function of genes in human cells. The lentiviral library of sgRNA can be used against the genes detected by functional screening for sensitive analyzes of next‐generation sequencing. The screening of this powerful library, with a loss of function approach, is expected to facilitate the identification of genes that play a major role in different bioprocesses, such as drug toxicity, targeted therapeutic molecules, and the expression of specific phenotypes (Zhou et al., 2014). 10 | SOME STUDIES CONDUCTED USING CRISPR–Cas9 SYSTEM The screening for 2368 genes via the CRISPR–Cas9 system in melanoma cells revealed a new targeted therapy for immunotherapy of cancer using PD‐1 inhibitors. In this study, the Cas9 protein was sustainably cloned initially within cancer cells. Then, gRNA was designed for 2368 different genes, each injected by a viral vector into its own cells. Eventually, the modified cells were grafted into rats to evaluate the effect of PD‐ 1 inhibitor. A new targeted therapy called Ptpn2 was identified in addition to the two mentioned types (PD‐L1 and CD47). The Ptpn2 gene deletion increased the intensity of the immune signal pathway that reduced tumor cell growth and ultimately cell death. Subsequent studies should determine the mechanism of action of Ptpn2 gene inhibitors in the treatment of cancer and whether this gene can be used as an efficient novel targeted therapy (Manguso et al., 2017). This study attempted to reveal the mechanism of this step in obtaining a new spacer sequence. At this stage, the Cas1–Cas2 proteins carry out the main role. Structural studies showed that, in addition to these two proteins, a third factor called integration host factor (IHF) is also involved in this mechanism. The IHF links to the start site of a new spacer and bends DNA to a U‐shaped structure that allows Cas1–Cas2 binding to both DNA segments. This finding indicates that, unlike other Cas proteins, both Cas1 and Cas2 proteins detect their target often through the spatial shape and other Cas proteins detect their sequences, such as PAM, at the target site. It was previously shown that these two proteins are capable of saving images and videos. Now, according to determine the mechanism of action of Cas1–Cas2, there is the prospect that other information, in addition to images and videos, can also be saved in CRISPR–Cas systems (Wright et al., 2017). The researchers used the CRISPR–Cas system and managed to save pixel‐based information of black and white images and short videos in the genome of a living bacterial cell population. In addition, the findings of this study on the performance of Cas1 and Cas2 proteins provide very useful information from the adaptation stage (Shipman, Nivala, Macklis, & Church, 2017). Viral anti‐CRISPR proteins have been shown to improve the function of the CRISPR–Cas system in genome editing. The use of AcrIIA4 inhibitory protein in cells edited by the CRISPR–Cas system has been about four times reduction in the off‐target occurrence rate without affecting the on‐target performance of this system. Such studies prove that the CRISPR–Cas editing system can be controlled in the cell and body and can become the perfect method to engineer the genome as accurately as possible (Shin et al., 2017). 11 | CRISPR–Cas9 TECHNOLOGY FOR INHIBITING RETINAL ANGIOGENESIS The vascular endothelial growth factor receptor 2 plays a major role in the angiogenesis process. Using the CRISPR–Cas9 system and the adeno‐associated virus (AAV) viral vector, researchers were able to prevent the angiogenesis in the clinical model for the first time, resulting in loss of vision. Further studies are needed to examine the safety and efficacy of this strategy in the treatment of angiogenesis‐ related disorders (Huang et al., 2017). Scientists used CRISPR technology to decode a rare and sometimes lethal childhood syndrome (dyskeratosis congenita), which causes the gradual loss of the ability to produce vital blood cells. Because the rat models of telomere deficiency are unable to show the full effects of this syndrome, the researchers through CRISPR technology managed to induce two mutations associated with the disease to human stem cells and observed that the cells developed short telomeres as the same as the disease. The inhibition of downstream effects of this deficiency showed no impact on the prevention of telomere short- ening, but the gradual disappearance of the blood cells was treated and the blood cells restarted to produce (Fok et al., 2017). A study was conducted to investigate the correction of a pathogenic gene mutation (MYBPC3) in a human embryo with hypertrophic cardiomyopathy (a disease that ultimately causes heart failure). In this study, it is interesting to note that the paternal mutated gene cleaved by the CRISPR–Cas9 is more effective replaced by maternal gene than synthesized through the DNA. The result of human embryonic manipulation was much more efficient than the manipulation of the induced pluripotent stem cells. The efficiency was 72.2% in the human embryo and 17.61–27.9% in stem cells, which indicates that the human embryo uses a DNA repair mechanism different from the somatic and stem cells. The injection of 5758 | KHADEMPAR ET AL. the Cas9 protein form for this test resulted in no off‐target in this strategy (Ma et al., 2017). In the treatment of many advanced cancers, the immunotherapy can be very effective in some patients but ineffective in some other patients. Researchers through the CRISPR–Cas9 system were able to detect 100 essential genes in the cell line of the melanoma whose presence is essential for the function of T cells. Deleting any of these genes showed that the cancer cells had more resistance to T cell. This study used the 2CT‐CRISPR technique in which T cells were considered as an effector and melanoma cells as a target (Patel et al., 2017). Using the RNA‐targeting Cas9 (RCas9) technique, the research- ers have been able to correct molecular mutations leading to microsatellite repeat expansion diseases, such as myotonic dystro- phies type 1 and type 2, hereditary common forms of ALS, and Huntington’s disease. In the RCas9 technique, instead of recruiting the engineered Cas9 to DNA, it binds to the target RNA. The efficacy of this technique is estimated at 95% or more in the treatment of microsatellite repeat expansion diseases. This study is important because of engineering a new form of CRISPR–Cas9 system. Moreover, this engineered system is easily presented to the target tissue by a viral vector because of deleting the regions of the engineered Cas9 protein sequence that is required to bind to DNA; as a result, the size of this engineering form is smaller than its wild type (Batra et al., 2017). Researchers used the CRISPR technology to produce less fatty pigs that have thermogenic capacity. The pigs are cold‐sensitive because of the absence of the UPC1 gene that is responsible for regulating body temperature in other mammals,especially when the air is cooled. Older pigs tolerate to some extent the cold temperatures due to more fat in the body. The researchers through the CRISPR–Cas9 system were able to link the rat UPC1 gene to the pig embryo. Grown pigs showed that they could withstand cold temperatures and had about 24% less fat in their body. After 6 months, the engineered pigs were killed to observe other changes, but no abnormality was observed and it was reported that all pigs had reproductive ability (Zheng et al., 2017). The target gene in this study was superoxide dismutase 1, which became knockout. The onset of the disease in the treated rat model was postponed for 5 weeks, and the rat survived 1 month longer than untreated rats. It cannot yet be said that the disease is treated, but the system is necessary to be optimized to target a greater percentage of cells (Gaj et al., 2017). 12 | IN VIVO ACTIVATION OF THE GENE EXPRESSION BY CRISPR–Cas9 SYSTEM In previous studies, dCas9 variant was always merged into an activator domain such as VP64. The limitation of this system is the large size of the chimeric sequence and it cannot be easily introduced by AAV to the targeted cell or animal model. The new idea has been proposed in this study, according to which: 1. Wild‐type Cas9 or dCas9 can be used to activate the target gene. 2. The Cas9 variant can be introduced to the system apart from the switcher and gRNA in a separate AAV, which has no longer spatial constraints for presentation (Liao et al., 2017). 13 | THE WILD ‐TYPE Cas9 TO ACTIVATE THE GENES In this study (Liao et al., 2017), it has been shown that if the gRNA sequence is 14–15 bp (known as dead gRNA or dgRNA), the Cas9 will be directed to the target site but will be unable to cause the DSB. In this study, the two MS2 domains (the SAM system) were used to reassemble the components. The MS2 domains recruit the MPH transcriptional activation complex. One of the advantages of this approach is indirect actions of epigenetic changes by recruiting a transcription machine, rather than being directly triggered by recruiting the epigenetic modulators. The method presented in this study has had promising results in the rat model for acute kidney failure, type I diabetes, and muscular dystrophy (Liao et al., 2017). 14 | CONCLUSION The discovery of CRISPR–Cas9 technology as a bacterial immune system against pathogens and its usage as an efficient tool for making targeted changes in the genome has led to a huge revolution in basic biology research. The growing ability of this technology is indis- putable in the systematic study of the gene function in mammalian cells, genomic variations during the progression of cancers and other diseases, and its potential for modifying genetic mutations causing genetic disorders. In this regard, evidence suggests that future studies have focused on optimizing this technology. Better under- standing of the mechanisms of intracellular repair systems following a DSB induced by Cas9 endonuclease improves the characteristics of targeted genomic changes. The development of special methods is required for the safe and efficient transfer of Cas9 protein and guide RNA to cells and tissues for the proper utilization of this technology in human gene therapy. Besides several advantages of CRISPR–Cas9 system, this system could be introduced to clinical applications in the fast and easy way. For example, several studies indicated that CRISPR–Cas9 system could be used as an effective approach for creating a new generation of chimeric antigen receptor T cells. However, there are still discussions about the safety and efficiency of CRISPR–Cas9 technology. One of the biggest concerns on this technology is the use of profitable individuals to make changes in human embryos in line with eugenic goals. Hence, there is a need for an accurate, responsible, and humanistic monitoring and control in this area by an authoritative international organization (Krishan, Kanchan, & Singh, 2016). 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