The DNA recombinant technology developed in the 1970s remarkable the new biology, enabling to engineer DNA molecules. This modern biotechnology established linkages between genetic variations and biological phenotypes (Ishino et al., 1987[80]; Mojica et al., 2000[130]; Hsu et al., 2014[78]). The traditional gene therapy based on vector-mediated overexpression methods was developed to supply the cells with the functional gene copies for monogenic recessive diseases (Hsu et al., 2014[78]). Currently, the gene therapy using the CRISPR-Cas9 system is suited for recessive and dominant inherited monogenic disorders (Jiang and Marraffini, 2015[85]; Kocher et al., 2017[96]; Sürün et al., 2018[166]).

Virtually, CRISPR-Cas9 can repair endogenous mutations and insert specific or random mutations (Pelletier et al., 2015[139]). In this sense, Cas9 may be used to correct single nucleotide polymorphism (SNP) where at least one copy of the functional gene can be expressed in its natural environment (Hsu et al., 2014[78]). If the monogenic disease is due to the duplication of genomic sequences, Cas9 may be exploited to delete the duplicated sequences. For the expansion of trinucleotide repeats two simultaneous DSBs may excise the repeat region (Hsu et al., 2014[78]). For monogenic dominant disorders, the NHEJ may inactivate the mutant allele, and the sgRNA will be designed targeting to the specific allele with the SNP (Hsu et al., 2014[78]). Rare diseases can also benefit from Cas9 technology (Pelletier et al., 2015[139]; Kocher et al., 2017[96]). Using this system is possible to prevent non-genomic elements as virus (Kwarteng et al., 2017[102]; Sakuma et al., 2016[150]; Pelletier et al., 2015[139]), by the promotion of mutation and repair NHEJ-mediated, circumventing infection without the need for continuing treatment (Kwarteng et al., 2017[102]; Pelletier et al., 2015[139]). The CRISPR-Cas9 multiplexed with the Cas9n vector can reduce viral replication (Sakuma et al., 2016[150]).

The perturbation of multiple genes simultaneously can also allow the discovery of new generation drugs and therapeutics, tested in the cell or transgenic animal created through CRISPR-Cas pathway (Jiang and Marraffini, 2015[85], Hsu et al., 2014[78], Pelletier et al., 2015[139], Kocher et al., 2017[96]). The animal models CRISPR-Cas9-based have been produced to overcome the delivery challenges and to provide tools for in vivo or ex vivo study of mutations associated with human diseases (Dai et al., 2018[43]; Erard et al., 2017[55]; Jiang and Marraffini, 2015[85]; Long et al., 2014[121]; Wangensteen et al., 2018[174]; Wu et al., 2013[181]; Markossian et al., 2018[127]; Hsu et al., 2014[78]; Platt et al., 2014[143]; Kocher et al., 2017[96]; Shi et al., 2017[160]). The most well-known objective considering CRISPR-Cas9 involves the generation of frameshift indels (insertion or deletion) of random mutations in single or multiple genes at once to better understand cellular physiology (Komor et al., 2017[97]; Markossian et al., 2018[127]; Billon et al., 2017[14]; Pelletier et al., 2015[139]; Jiang and Marraffini, 2015[85]). The development of a multi-color and multi-locus Cas9 proteins or sgRNAs by labeling with fluorescent molecules can be an alternative method to the traditional probe DNA labeling, providing living cell imaging about complex chromosomal architecture and nuclear organization (Jiang and Marraffini, 2015[85]; Liu et al., 2017[118]; Ye et al., 2017[187]; Dominguez et al., 2016[47]; Knight et al., 2018[94]; Hsu et al., 2014[78]).

Genome editing can promote either chromosomal biallelic or monoallelic frameshift mutations (Platt et al., 2014[143]). Chromosomal alterations are efficiently generated by large insertions or deletions that disrupt intra or intergenic regions by DSBs (Zuo et al., 2017[197]; Pelletier et al., 2015[139]). Moreover, chromosomal translocation can also be produced using an HDR oligonucleotide with sequence homology to both chromosomal locus and co-injected with CRISPR-Cas9 (Pelletier et al., 2015[139]).

In the central dogma of molecular biology is the combined control of gene expression coordinating the events of gene transcription and translation, as the turnover of RNAs and proteins. The dysregulation of gene expression often results in cancer, metabolic disorders, cardiovascular and other human diseases (Komor et al., 2017[97]; La Russa and Qi, 2015[103]; Chen and Qi, 2017[29]). Significant efforts have been devoted to engineering transcriptional factors to control biological processes and, thereby, create new therapeutic strategies (Gilbert et al., 2013[66]).

The regulatory strategies using dCas9 can produce levels of gene control without precedent (La Russa and Qi, 2015[103]). The control of the transcriptional regulation is crucial for understanding cellular behavior and dCas9 targeting regions upstream or downstream of the transcription start site modulates cellular response (Braun et al., 2016[17]). Regulation of gene expression by CRISPR-dCas9 system is termed as CRISPRa for gene activation or CRISPRi for gene repression (Komor et al., 2017[97]; Gilbert et al., 2013[66]; La Russa and Qi, 2015[103]; Perez-Pinera et al., 2013[141]; Chen and Qi, 2017[29]), becoming versatile when multiple sgRNAs/dCas9 fusions are multiplexed to regulate multiple targets within the same pathway (La Russa and Qi, 2015[103]). For example, dCas9 may be employed as a transporter protein directed to specific or multiple loci guided by sgRNA, activating or repressing gene expression (CRISPRa/i) (Komor et al., 2017[97]; Chen and Qi, 2017[29]; La Russa and Qi, 2015[103]; Perez-Pinera et al., 2013[141]).

However, it is noteworthy that genome organization in eukaryotes is complex and DNA regulatory elements can be far from the transcriptional start point. Epigenome also coordinates gene expression, and chromatin remodeling and chemical modifications of histones must be taken into account for expression regulation (Gilbert et al., 2013[66]). Three-dimensional organization of chromatins exerts a putative role in gene expression, and how chromatin loops are involved in this process needs to be clarified. In this field, CRISPR/dCas9 may interfere in nuclear architecture demonstrating the power of this system to reorganize chromatin loops (Morgan et al., 2017[131]).

The gene expression by epigenome editing can be regulated by dCas9:sgRNA complex using combinations of synthetic transcription factors to bind near the transcriptional endogenous human gene promoters with a relevant role in medicine (Komor et al., 2017[97]; Cheng et al., 2013[31]; Perez-Pinera et al., 2013[141]; Polstein and Gersbach, 2015[144]). The co-recruitment of multiple activators may be the strategies to produce the second generation of CRISPRa (La Russa and Qi, 2015[103]). The transcriptional activation domain VP64 from Herpes Simplex Viral Protein 16, fused with dCas9 (dCas9-VP64) was used to modulate the expression of a wide gene (Komor et al., 2017[97]; Guo et al., 2017[72]; Balboa et al., 2015[10]; Perez-Pinera et al., 2013[141]). The dCas9 fused to a histone acetyltransferase stabilized the expression of a transcription factor regulated by epigenetic modification in inflammatory conditions modulating cellular response (Okada et al., 2017[136]).

Alternatively, CRISPRi is an approach for perturbing gene expression with selectivity to RNA polymerase, transcription elongation and/or with the sgRNA binding to specific DNA targeting the protein-coding region on a genome-wide scale (Jiang and Marraffini, 2015[85]; Chen and Qi, 2017[29]). The use of CRISPRi represents an exciting field to the counterpart RNA interference (RNAi) (Jiang and Marraffini, 2015[85]; La Russa and Qi, 2015[103]; Larson et al., 2013[105]). The use of quantitative fluorescence assays and native elongating transcript sequencing was described to produce a protocol for gene silencing at the transcriptional level through sgRNA providing the repression activity of CRISPRi as a complementary approach to RNAi (Larson et al., 2013[105]).

The advantage and disadvantages of the use of RNAi and CRISPRi need to be systematically evaluated about the effectivity in each different type of cell. In the CRISPRi system, the bioinformatics modeling identifying high-confidence hits to generate sgRNAs directed to the target and testing large pools is important to find the ideal sgRNAs reducing off-targets (Erard et al., 2017[55]; La Russa and Qi, 2015[103]). The success of the CRISPR-Cas/dCas system has empowering researchers to better understand prokaryotic and eukaryotic genome functions and biology system (Jiang and Marraffini, 2015[85]; Hsu et al., 2014[78]).

Obesity affects nearly 500 million people worldwide and is at risk of developing cardiovascular disorders, cancer, and type 2 diabetes (Garcίa-Jiménez et al., 2016[63]). Obesity is primarily due to a positive energy balance, whereby the energy consumption exceeds expenditure, leading to energy storage, predominantly as lipids in white adipocytes (Claussnitzer et al., 2015[37]). Notably, severely obese patients had medical costs that were $1980 higher annually per capita, compared to that of individuals within a normal body mass index (BMI) (Julie and Jacob, 2015[90]). BMI comprised of strong genetic components (40 to 80 % heritability) with several genes expressed in the hypothalamus and plays a crucial role in appetite regulation (Locke et al., 2015[120]).

Investigators at MIT, Havard University, and the Broad Institute of Cambridge had identified a variant of the fat mass and obesity-associated (FTO) gene that is responsible for the development of fat cells in the body and thus it is considered as one of the primary factors for weight gain (Claussnitzer et al., 2015[37]). However, what is worth to mention here was not the isolation of the FTO gene variant, the researchers were able to modify the living cells in mice that carrying variant using CRISPR-Cas9 (Maclean, 2016[123]).

Using DNA editing tools, they have identified a genetic switch that facilitates the regulation of body metabolism. The switch governs whether common fat cells burn energy or store it as fat (Vogel, 2015[170]). CRISPR/Cas9 also can be used to convert the obesity-promoting FTO gene in adipocyte precursor cells. The treated cells had lowered IRX3 and IRX5 expression, and ultimately spun up the energy-burning machinery (Claussnitzer et al., 2015[37]).

In another study, Yue et al. (2017[189]) reported that CRISPR-mediated genome editing controlled the release of glucagon-like peptide 1 (GLP1), a crucial incretin that regulates blood glucose homeostasis. In this study, GLP-1 induction from engineered mouse cells grafted onto immunocompetent hosts would be triggered by the administration of the antibiotic doxycycline, which subsequently reduces the appetite and decreases the high fat diet-induced obesity. Importantly, GLP-1 induction can reverse the high-fat diet-induced weight gain. Therefore, the ability in the manipulation of the uncovered pathway, such as overexpression or knockdown of the upstream regulator ARID5B, a genome editing of the predicted causal variant rs1421085, and overexpression or knockdown of target genes IRX5 and IRX3, had a prominent effect on obesity phenotypes.

As a consequence of the global rise of obesity, the current and future burden of cancers associated with obesity tends to increase. This is not only affecting the occurrence of cancer, a high prevalence of obesity also affects prognosis among cancer survivors (Arnold et al., 2016[7]). Cancer is a disease featured by multiple epigenetic and genetic alterations in tumor suppressor genes and oncogenes (Deben et al., 2016[45]). All cancers exert stepwise mutations that allow the cells to multiply and exhibit their malignancy features (Economides et al., 2017[52]; Xu et al., 2017[182]). Cancer is genetically complex with hundreds of translocations, mutations, and chromosome losses and gains per tumor (Greer et al., 2017[70]). Inappropriate activation of Wnt pathway has linked with different types of cancers. Loss of the adenomatous polyposis coli (APC) gene function induces a premalignant precursor lesion, which is a hallmark of human colorectal cancers (CRC) (Novellasdemunt et al., 2015[135]). Given the advance in comprehensive structural characterization in cancer genomes and sequencing technology (Vogelstein et al., 2013[171]), the mutations in the Wnt pathway frequently implicated in human cancers (Duchartre et al., 2016[50]). Although the fact that most of the pathway components have been characterized, the function of Wnt pathway within the context of cancer biology is intriguingly complex and yet fully understood (Zhan et al., 2017[192]).

Over the past decades, research and development on genome engineering technologies have made it possible to modify or precisely delete specific DNA sequences in the genome of the cells or animal models (Sánchez-Rivera and Jacks, 2015[152]). The ability to correct such mutations is a primary goal in cancer treatment. There is substantive evidence based on the role of genome editing with CRISPR/Cas9 as a rapid genetic manipulation approach in any genomic locus (Dow et al., 2015[49]).

It is also demonstrated that CRISPR/ Cas9 system has been used to generate cancer cells from mouse primary cells or in vivo tissue (Heckl et al., 2014[76]; Xue et al., 2014[183]). Even though the requirement of Wnt-β-catenin signaling axis in RNF43-mutated pancreatic ductal adenocarcinoma (PDAC) (Jiang et al., 2013[86]), the genetic complexity in this pathway for example co-receptors and intracellular signaling components, frizzled (FZD) receptors and Wnt ligands made the prediction of this therapeutic targets become difficult. Therefore, identification of this specific genetic vulnerability using genome-wide CRISPR-based genetic screens may provide an unbiased and powerful means to identify context-specific fitness genes that can be harnessed in the treatment of cancer. Genome screening using CRISPR/Cas9 enables the detection of the Wnt-FZD5 signaling circuit in an RNF43-mutant pancreatic tumor (Steinhart et al., 2017[164]).

Treating de novo Kaposi's sarcoma-associated herpesvirus (KSHV)-infected endothelial cells with epilipoxinand lipoxinexhibits an anti-inflammatory effect by reducing the nuclear factor kappa B(NF-κB), extracellular signal-regulated kinase ½ (ERK1/2), cyclooxygenase-2(COX-2), 5-lipoxygenase, and Akt expression. Furthermore, treatment with lipoxinusing CRISPR/ Cas9 technology-mediated lipoxin A4 receptor/formylpeptidyl receptor (ALX/FPR) gene deletion implied the efficiency of lipoxin receptor ALX in lipoxin signaling (Chandrasekharan et al., 2016[26]). In this study, a viral miRNA cluster has been identified as an important factor in the downregulation of lipoxin A4 secretion in the host cells (Chandrasekharan et al., 2016[26]). Additionally, De Ravin et al. (2017[44]) also demonstrated that CRISPR/Cas9 could repair a mutation in the CYBB gene of CD34+ hematopoietic stem and progenitor cells (HSPCs) for those of the patients with immunodeficiency disorder X-linked chronic granulomatous disease (X-CGD). The beta-hemoglobinopathies, such as beta-thalassemia and sickle cell disease are caused by beta-globin (HBB) gene mutations which had affected millions of people worldwide. Surprisingly, using CRISPR-based methodology the ex vivo gene can be corrected in patient-originated hematopoietic stem cells after autologous transplantation.

The miRNAs play a crucial role in the development of breast cancer. An earlier study has reported that miRNAs expression was changed significantly in cancer cell lines and tumor tissues (Ji et al., 2017[83]). miRNA dysfunction is a biomarker in various pathological diseases such as cancer (Ji et al., 2017[83]). Therefore, miRNA has been recognized as a robust biomarker in ovarian, prostate, and ovarian cancers for therapeutic targeting (Smith et al., 2017[161]). This potential therapeutic effect has been demonstrated by Abdollah et al. (2017[1]), who found that the expression of miR-130a-5p was downregulated in breast cancer (MCF7) cell line using CRISPR silencing system. It was also revealed from several in vitro and in vivo studies that this editing tool has potential in inhibition of cancer cell growth and increases cell apoptosis (Liu et al., 2014[119]; Zhen et al., 2017[195]). Importantly, CRISPR systems are being applied to ameliorate genetic disorders in animals and seem to be employed in human diseases (Barrangou and Doudna, 2016[11]). The prospects of this gene-editing technology trigger a biomedical duel in China when the CRISPR-Cas9 technique was first introduced to a patient with aggressive lung cancer at West China Hospital, Chengdu (Cyranoski, 2016[42]).

In addition to the significant therapeutic approach of in vivo somatic genome editing to alter genetic defects (Long et al., 2014[121]; Yin et al., 2014[188]), its applications in cancer modeling have been convincingly reported (Table 1(Tab. 1); References in Table 1: Aubrey et al., 2015[8]; Blasco et al., 2014[15]; Chiou et al., 2015[33]; Dow et al., 2015[49]; Xue et al., 2014[183]; Zuckermann et al., 2015[196]). For instance, several groups have demonstrated that direct in vivo delivery of the relevant gRNA and Cas9 to the specific tissue using viral vectors or naked DNA can be used to initiate tumorigenesis and suppress somatically tumor-suppressor genes (Platt et al., 2014[143]; Xue et al., 2014[183]; Chiou et al., 2015[33]; Zuckermann et al., 2015[196]). This approach has been successfully utilized to inactivate the tumor suppressor genes such as liver kinase B1 (Lkb1), transformation-related protein 53 (Tp53), phosphatase and tensin homolog (Pten), and adenomatous polyposis coli (APC) in the lungs of adult mice (Platt et al., 2014[143]). Preclinical findings from in vitro and in vivo studies on mechanisms involved in cancer suggest that the CRISPR-Cas9 technique could modulate pathway involving in cancers. Targeting the cancer cells nucleotide sequences with the cellular genome is an attractive approach. Collectively, the ability to correct the cancer-associated mutations has been identified as a powerful treatment option. These techniques facilitate in the correction of mutations in the cancer cells.

Diabetes is a chronic and metabolic disease featured by elevation of blood glucose levels, which cause serious damage to the blood vessels, eyes, heart, nerves, and kidneys (World Health Organization, 2017[179]). According to the World Health Organization (2017[179]), there are 422 million adults having diabetes with 1.6 million deaths annually (World Health Organization, 2017[179]). CRISPR/Cas9 could alter the gene that is responsible for encoding a hormone known as glucagon-like peptide-1 (GLP-1). GLP-1 promotes the release of insulin and facilitates in the reduction of glucose from the blood (Yue et al., 2017[189]). This therapeutic potential was not only exhibited in in vivo genome editing, its applications in Type 1 diabetes in a mouse modeling have also been demonstrated. For example, Ptpn22^R619W mice showed an increase in insulin autoantibodies and high susceptibility of Type 1 diabetes (Lin et al., 2016[114]). Notably, inhibition of FOS/JUN pathway either through CRISPR-mediated suppression of FOS rescued the inability of CDKAL1 null cells to alter glycemia in streptozotocin-diabetic mice (Rutter, 2016[149]). Taken together, these editing techniques hold great promise in future use in the treatment of diabetes.

Cardiovascular diseases (CVDs) are a major health problem and the leading cause of death globally (Go et al., 2013[68]). Several polymorphisms have already been identified and implicated in the pathogenesis of CVDs and more continue to be located by scientists every day (Fiatal and Ádány, 2018[56]). The emergence of gene-surgery technologies has given researchers new approaches to study and treat the common complex CVDs such as inherited cardiomyopathies, valvular diseases, primary arrhythmogenic conditions, congenital heart syndromes, hypercholesterolemia and atherosclerotic heart disease, hypertensive syndromes, and heart failure with preserved/reduced ejection fraction (Pasipoularides, 2018[137]).

The three universally used tools are Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas9 (CRISPR/Cas9) system (Termglinchan et al., 2016[168]). Each system has its own advantages and limitations and has been applied in various scientific fields to increase the understanding of normal gene regulation and the pathophysiology of the disease, as well as to facilitate the development of novel therapies (Seeger et al., 2017[154]).

One of the major goals of gene surgery is replacing defective, down-regulated, or missing genes with normal, enabling the normal function or, alternatively, adds a new gene to restore or improve well-being. In fact, there are different approaches for using gene editing according to two categories of CVDs: diseases caused by primary cardiomyopathy that could be corrected by genome editing within the cardiac muscle, and diseases secondarily caused by extracardiac influences (Strong and Musunuru, 2017[165]). In the first category, the most likely example are the inherited cardiomyopathies, such as familial hypertrophic cardiomyopathy and dilated cardiomyopathy. In a large proportion of patients, these cardiomyopathies are caused by single dominant mutations within sarcomere genes (Musunuru, 2017[133]). In principle, these diseases could be prevented or arrested by two different strategies: correction of the mutant allele by homology-directed repair (HDR), or allele-specific gene disruption via Non-Homologous End-Joining (NHEJ) strategy, removing the dominant influence of the mutant allele (Jiang et al., 2013[84][86]).

The first strategy might be extremely challenging to achieve owing to limited HDR in adult cardiomyocytes. An approach similar to the second strategy has been achieved in a mouse model through the use of RNA interference, suggesting the viability of the NHEJ genome-editing approach (Coelho et al., 2013[38]). The success of the NHEJ approach would depend on being able to achieve stringent specificity for the mutant allele, which usually differs from the wild-type allele by only a single base pair. For CRISPR/Cas9, this specificity would be most feasible if the causal mutation created a point accepted mutation (PAM) site that was not present in the wild-type allele (Chong et al., 2014[35]).

The second category of diseases includes the treatment of hypercholesterolemia to reduce the risk of myocardial infarction, via the disruption of proprotein convertase subtilisin/kexin type 9 (PCSK9) or other lipoprotein regulators in the liver (Cai et al., 2016[18]). When PCSK9 is dysregulated, such as through gain-of-function mutations in the PCSK9 gene, the protein impairs low-density lipoprotein (LDL)-cholesterol clearance by acting as an antagonist to the LDL receptor, thereby promoting hypercholesterolemia. Conversely, naturally occurring loss-of-function mutations reduce the risk of myocardial infarction by markedly reducing blood cholesterol levels (Chadwick and Musunuru, 2017[24]). Due to this observation, two antibody-based therapies targeting PCSK9 have been developed and recently approved; however, these therapies must be delivered by injection every few weeks (Ito and Santos, 2017[81]). Another example is cardiac amyloidosis, in which production and secretion of a mutant form of the transthyretin (TTR) protein by the liver into the bloodstream results in the accumulation of the mutant protein within the cardiac muscle. Disruption of the TTR gene in the liver by genome editing might arrest or even reverse the cardiomyopathy, a goal currently being pursued with RNA interference (Coelho et al., 2013[38]).

A distinct approach to leveraging genome editing for the treatment of cardiovascular disorders would be to perform the editing ex vivo. This might entail generating induced pluripotent stem cells (iPSCs) from a patient, editing the iPSCs so as to correct a mutation or otherwise render the cells protective against disease, differentiating the edited iPSC into the desired cell type (such as cardiomyocytes), and transplanting the differentiated cells back into the patient (Wu et al., 2018[180]). Even though this approach remains largely theoretical for CVDs. Currently, efforts are directed toward complex, polygenic, chronic cardiac diseases, such as acute heart disease (AHD) and heart failure (HF) (Lim, 2017[112]).

The application of the gene surgery tools in CVDs presents a novel and rapidly advancing technology with promising results. However, significant challenges remain, including enhancing specificity and minimizing off-target effects, increasing efficiency, and improving the selection of targeted sites and delivery methods, and especially for in vivo genome engineering. Further refinements are needed to fully exploit the potential of genome editing to be a vital tool for future precision medicine treatment for CVDs.