|Year : 2023 | Volume
| Issue : 4 | Page : 201-207
A new era of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 gene editing technology in cardiovascular diseases: Opportunities, challenges, and perspectives
Kumar Rahul1, Sushil Kumar Singh1, Sarvesh Kumar1, Vivek Tewarson1, Mohammad Zeeshan Hakim1, Karan Kaushik2, Satish Kumar3, Bhupendra Kumar1
1 Department of Cardiovascular and Thoracic Surgery, King George's Medical University, Lucknow, Uttar Pradesh, India
2 Department of Cardiac Anaesthesiology, King George's Medical University, Lucknow, Uttar Pradesh, India
3 Department of Internal Medicine, King George's Medical University, Lucknow, Uttar Pradesh, India
|Date of Submission||04-May-2023|
|Date of Acceptance||11-Sep-2023|
|Date of Web Publication||03-Nov-2023|
Dr. Kumar Rahul
Department of Cardiovascular and Thoracic Surgery, King George's Medical University, Shahmina Road, Lucknow - 226 003, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Cardiovascular diseases (CVDs) remain major causes of global mortality in the world. Genetic approaches have succeeded in the discovery of the molecular basis of an increasing number of cardiac diseases. Genome-editing strategies are one of the most effective methods for assisting therapeutic approaches. Potential therapeutic methods of correcting disease-causing mutations or of knocking out specific genes as approaches for the prevention of CVDs have gained substantial attention using genome-editing techniques. Recently, the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has become the most widely used genome-editing technology in molecular biology due to its benefits such as simple design, high efficiency, good repeatability, short cycle, and cost-effectiveness. In the present review, we discuss the possibilities of applying the CRISPR/Cas9 genome-editing tool in the CVDs.
Keywords: Cardiovascular diseases, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9, genome editing
|How to cite this article:|
Rahul K, Singh SK, Kumar S, Tewarson V, Hakim MZ, Kaushik K, Kumar S, Kumar B. A new era of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 gene editing technology in cardiovascular diseases: Opportunities, challenges, and perspectives. Heart Views 2023;24:201-7
|How to cite this URL:|
Rahul K, Singh SK, Kumar S, Tewarson V, Hakim MZ, Kaushik K, Kumar S, Kumar B. A new era of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 gene editing technology in cardiovascular diseases: Opportunities, challenges, and perspectives. Heart Views [serial online] 2023 [cited 2023 Dec 6];24:201-7. Available from: https://www.heartviews.org/text.asp?2023/24/4/201/389344
| Introduction|| |
Cardiovascular diseases (CVDs) remain major causes of global mortality and one of the most serious health problems in the world. The global prevalence of CVDs has risen by 93% over the past three decades (from 271 million in 1990 to 523 million in 2019). Moreover, the total number of deaths due to CVDs has increased by about 54%, representing about one-third of all global deaths. It was demonstrated that CVD would be responsible for more than 23 million deaths (about 30.5%) by 2030 worldwide.,
CVDs include cerebrovascular disease (stroke), heart failure, hypertensive heart disease, rheumatic heart disease, peripheral arterial disease, cardiomyopathy, and several other cardiac problems. Several risk factors related to the development of CVDs such as lifestyle habits and environmental factors have been identified; however, these explain only a fraction of the events. Therefore, exploration of the underlying molecular mechanisms is important for explaining cases that are not related to known risk factors for the development of CVDs.
It has been proven that genetic predisposition plays a pivotal role in the development of CVDs. Genetic approaches have succeeded in the discovery of the molecular basis of an increasing number of cardiac diseases., In addition to the genes with known action in the cardiovascular system, the exploration of new genes associated with heart diseases may provide novel therapeutic strategies for CVDs.
Genome-editing strategies are one of the most effective methods for assisting therapeutic approaches. Potential therapeutic methods of correcting disease-causing mutations or of knocking out specific genes as approaches for the prevention of CVDs have gained substantial attention using genome-editing techniques.,, Videlicet, gain-of-function mutations in the proprotein convertase subtilisin-like kexin type 9 (PCSK9) gene, which is a major regulator of low-density lipoprotein (LDL) receptor levels and LDL-cholesterol (LDL-C) concentrations, has been reported to increase LDL-C levels, leading to an increased risk of hypercholesterolemia and coronary heart disease (CHD).
In contrast, studies on loss-of-function mutations in PCSK9 indicate that inactivation of PCSK9 lowers LDL-C levels and reduces CHD, suggesting PCSK9 inhibition as a valid therapeutic method in the management of hypercholesterolemia and related diseases.
Since 1996, two kinds of designed nucleases including zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been developed. They acted as the first and second generations of gene-editing technology, respectively. Nevertheless, the high cost, low efficiency, and limited accessibility have limited the application of these tools. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, which was first described in 2012 by Jinek et al., was developed as the third generation of gene-editing technology, and it has become the most widely used genome-editing tool in molecular biology because of its benefits such as simple design, high efficiency, good repeatability, short cycle, and cost-effectiveness.,,
In the present review, we focus on the possibilities of applying the CRISPR/Cas9 genome-editing tool in CVDs.
| Gene-Editing Mechanisms|| |
After finding endonuclease restriction enzymes, researchers use these enzymes for different purposes in research. However, some exciting experiments, such as genome manipulation, mutation modification, and deletion of specific genes, have always been the focus of scientists. This passion led to the development of various methods for genetic manipulation at the molecular level and gene-editing technology.
| Zinc-Finger Nuclease Gene-Editing Mechanism|| |
One of the methods was the ZFN method. Zinc-finger transcription factors or ZF-TFs are a set of designed and engineered proteins that can attach to a specific part of DNA in a completely specific way. The protein was discovered in the study of Xenopus oocytes in 1985. ZFNs consist of two parts: the first part is zinc-finger DNA-binding domains that can attach to a specific sequence of DNA. Another part is an engineered nuclease called Fok1. These two domains fuse to form a complex that can detect a specific sequence in DNA and attach to it, using its enzymatic domain to cleavage DNA. These two domains combine to form a complex that can detect a specific sequence in DNA attached to it and use its enzymatic domain to cleavage DNA.
Three factors affect the characteristics of ZFNs; the amino acid sequence that makes up each zinc finger, the number of fingers that are components of the complex, and the integration of the nuclease domain. Despite the advantages of this method, some of the factors and disadvantages of this method created limitations for the use of this method on a large scale and encouraged scientists to find alternative methods such as the high cost, time-consuming optimization of this method, and limitations in selecting target locations.
| Transcription Activator-Like Effector Nucleases Gene-Editing Mechanism|| |
Like zinc fingers, TALENs are made up of two different parts, one for identifying the target site on DNA and the other for the nuclease enzyme. The domain to identify the junction on the DNA in this method is TAL effector DNA binding, which can be designed and engineered, and another domain is an enzyme called Fok1. The TALE part is a protein that binds to the desired location on the DNA with 33–35 amino acids. This part, depending on the type of amino acids that make it up, can identify and bind to a specific part of the DNA.
TALENs have better efficiency and characteristics than finger zinc. However, despite the positive features of this method, some factors limit the use of this method as well, including being time-consuming and the requirement of a 5'thymine base in the target sequence.
| Clustered Regularly Interspaced Short Palindromic Repeats/Crispr-Associated Protein 9 Gene-Editing Mechanism|| |
CRISPR/Cas9 technology consists of two parts called guide RNA (gRNA) and Cas9 enzyme. gRNA consists of approximately 20 nucleotides that fit into a larger RNA framework. This larger RNA framework is located on the target DNA and puts the Cas9 enzyme in the right position on the DNA. The gRNA is designed as complementary to target any locus in the genome and can easily be designed by design tools including https://wge.stemcell.sanger.ac.uk/, https://www.atum.bio/eCommerce/cas9/input, http://biotools.nubic.northwestern.edu/OligoCalc.html, and http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi.
When this gRNA is added to the Cas9 complex, it binds to the desired region on the DNA, making the Cas9 cut point coincide with the desired point. In this way, the cut is made from the target point on the DNA. This method is shown step by step in part (C) of [Figure 1]. The flowchart of CRISPR/Cas9 genome editing is presented in [Figure 2].
|Figure 1: Different gene-editing technologies. (a) Zinc-finger nucleases contain a chain of zinc-finger proteins that can bind to a specific sequence of DNA in a very specific site and, by complexing with a bacterial nuclease (Fok1), can cleavage DNA strands, (b) Transcription activator-like effectors can be designed to bind to the target DNA sequence, and this combination can cleave DNA from this specific site in association with the Fok1 nuclease enzyme, (c) Gene modification with clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method is currently one of the strongest and best tools for gene change at the level of nucleotide sequences in DNA structure. In this figure, the mechanism of the CRISPR/Cas9 method in gene editing is shown step by step. ZF: Zinc-finger, ZFN: ZF nuclease, TALE: Transcription activator-like effector, TALEN: TALE nucleases, CRISPR: Clustered regularly interspaced short palindromic repeats, Cas9: CRISPR-associated protein 9, gRNA: Guide RNA|
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|Figure 2: Flowchart of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 genome editing. Cas9: CRISPR-associated protein 9, gRNA: Guide RNA|
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The same as other techniques, this method has some advantages and disadvantages. Both ZFN and TALEN have the gene-editing ability, but CRISPR/Cas9 has several key advantages such as high efficiency, no drug selection required, easy delivery, and successful editing in different cell types. On the other hand, off-target cleavage is possibly more frequent in CRISPR/Cas9 than in TALENs and ZFNs.
Studies show that the CRISPR/Cas9 method is more efficient than other gene-editing methods. While the efficiency of CRISPR/Cas9 is 0%–81%, the efficiency of the TALEN method is 0%–76% and 0%–12% for ZFN. On the other hand, the possible target site is 500 bp and 36 bp for ZFN and TALEN, respectively, while 8 bp for CRISPR/Cas9. Furthermore, the TALEN and ZFN methods are sensitive to methylation, whereas the CRISPR/Cas9 method is nonsensitive. However, the CRISPR/Cas9 method has more potential off-target effects than TALEN and ZFN methods. In this respect, the ZFN method has at least off-target effects.
| Basic Studies and Clinical Findings|| |
In vitro models for clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9
In vitro investigations are used broadly because they are relatively easy to maintain and manage including simplicity, species specificity, and convenience-induced pluripotent stem cells (iPSCs) due to their close similarity to embryonic. Stem cells are the most appropriate model for assessing cardiomyogenesis in human cells. Numerous investigations have been done in the field of CVD with patient-specific human iPSCs (hiPSCs). These cells can be reprogrammed and differentiated into a diversity of cells for more functional analysis. More details in the realm of cardiomyopathy, including Barth syndrome (a mitochondrial dysfunction disorder caused by mutations in the tafazzin gene), have been identified using ZFNs, TALEN technology, or CRISPR/Cas9 gene-editing strategy.,
Zhang et al. showed that CRISPR/Cas9 ablation of special microRNAs disclosed their efficacies during the differentiation of mouse embryonic stem cells. MiRNA106a, miR17, and miR93 target the cardiac suppressor gene Fog2. Fog2 is a multi-zinc-finger protein, which is associated with a cardiac transcription factor, GATA-4. GATA-4 is required for normal heart development as well as hypertrophic responses in cardiac myocytes. In a human in vitro cardiac model, researchers suggested that KCNQ1-SupRep gene therapy by CRISPR-Cas9 in iPSC-derived cardiomyocytes (iPSC-CMs) can be considered for complete correction of long QT syndrome (LQTS). Similarly, Yamamoto et al., using the Cas9 double-nickase system in hiPSCs, generate in vitro allele-specific knockout models of channelopathy LQTS. In general, based on in vitro evidence, CRISPR/Cas9 shows great potency for future applications in vivo and human studies.
In vivo models for clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9
CRISPR/Cas9 has been applied to different small animals including zebra fish, rats, mice, and large animals such as pigs. Some mutations that are responsible for cardiomyopathies including dilated cardiomyopathy, Barth syndrome, LQTS, hypertrophic cardiomyopathy, and Duchenne muscular dystrophy (DMD) have been corrected by genome editing in patient-specific iPSC-CMs.,,
Drug resistance remains a challenge in the treatment of proprotein convertase subtilisin/kexin type 9 (PCSK9) 2-overexpressed low density lipid (LDL). Using CRISPR/Cas9 genome editing, Ding et al. reported a loss of function for the PCSK9 gene in the livers of mice and consequently a decrease in the cholesterol levels by over 40%. In another study, inhibiting several gene functions including apolipoprotein E, cluster of differentiation 36, LDL receptor, leptin, and ryanodine receptor type 2 (RYR2) using RNA-guided Cas9 nucleases were represented. A mice model of DMD enhanced skeletal muscle function 4 weeks after IM-adeno-associated virus-9 (AAV9)-Cas9 injection.
Furthermore, Mendell and Rodino-Klapac have shown that CRISPR/Cas9-treated mice significantly improved muscle function through clavation, repairing, or removing faulty exons of the dystrophin gene. Dysfunctional RYR2 is responsible for approximately 60% of all catecholaminergic polymorphic lethal ventricular tachycardia. In an animal model, researchers indicated that AAV serotype 9-based delivery of the Cas9 system can efficiently edit CMs by specifically targeting the disease-causing allele. Interestingly, other in vivo models indicated that AAV-CRISPR/Cas9-mediated Ldlr gene correction can ameliorate atherosclerosis phenotypes and be a potent treatment strategy for patients with familial hypercholesterolemia.
Overall, based on the results of the in vivo studies, CRISPR/Cas9-mediated gene editing system is a promising strategy to alter the function of genes connected to CVDs.
Prospective application for clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 in clinical human studies
CRISPR genome-editing technology seems to be worth watching for both researchers and clinicians. Human CRISPR/Cas9 clinical trials received ethical approval in China and the United States. Recent evidence claims that this strategy is a novel treatment for several genetic disorders, including some cancers, neurodegenerative diseases, sickle cell anemia, DMD, viral infections, immune disorders, cystic fibrosis, and CVDs.,,, Various studies have supported this hypothesis that the combination of genome-wide association studies and CRISPR genome-engineering strategy could play an important role in the development of human personalized medicine.
In a recent trial, researchers investigated the use of CRISPR/Cas9-based gene editing for treating two patients with inherited diseases: one patient with transfusion-dependent β-thalassemia (TDT) and the other in a patient with sickle cell disease (SCD). Based on their results, both patients had early, substantial, and sustained increases in fetal hemoglobin levels after the administration of CTX001, with more than 99% pancellularity during 12 months. Along with the reported advantages, some adverse events were documented in both patients such as pneumonia in the presence of neutropenia, sepsis in the presence of neutropenia, cholelithiasis, veno-occlusive liver disease with sinusoidal obstruction syndrome, and abdominal pain after TX001 infusion. Next step, the administration of CTX001 to an additional eight patients (six with TDT and two with SCD) was done. Their results supported further experimental testing of CRISPR/Cas9 gene-editing approaches for treating genetic diseases. The use of CRISPR/Cas9 gene-editing technology in clinical trials for the treatment of CVD is shown schematically in [Figure 3].
|Figure 3: Use of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene-editing technology in clinical trials for the treatment of cardiovascular disease (a) Sample is taken from a patient with cardiovascular disease, (b) Cardiovascular cells are isolated from the patient, (c) Corrections are made in cells with the CRISPR/Cas9 method, (d) The patient-specific corrected cells are transplanted into the patient. CRISPR: Clustered regularly interspaced short palindromic repeats, Cas9: CRISPR-associated protein 9|
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Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9: Future perspectives, concerns, and its application in heart disease
It is a fact that this method has promising potential for treating diseases. CVDs caused by a genomic defect constitute potential candidates for treatment with this method. However, research with this method is limited to in vitro and animal models only. If these researches continue, treatment methods at the gene level for different heart diseases will likely emerge. However, these experiments were limited due to some ethical problems. Can editing human fetal cells at the genome level be ethical? This issue is open to discussion. However, it is a fact that some may use this technology for nontherapeutic purposes, and this necessitates the ethical use of this technology.
| Conclusion|| |
Collectively, recent structural and mechanistic studies on the realm of CRISPR/Cas9 genome-editing technology in vitro, in vivo, and human studies open new therapeutic perspectives for treating CVDs. However, for the broad use of this method for human studies, some points need to be considered. First, since the SpCas9 and SaCas9 proteins are the most commonly used Cas9 proteins, the major delivery challenge in terms of packaging into AAV due to their large size must be resolved based on this fact. Discovering smaller Cas9 orthologs or reducing the size of the SpCas9 and SaCas9 proteins can be a strong point for solving this limitation. Furthermore, more characterization and optimization are needed for its therapeutic application. Beyond that, despite the suggestive application of CRISPR technology including genome editing, endogenous gene expression, epigenome editing, and edition of RNA, several challenges should be targeted in future studies.
In total, the unconquerable limitation of the CRISPR/Cas9 editing system is the variability in its efficiency and potential off-target gene editing. In addition, germline editing by this technique, mainly in humans, raises societal and ethical considerations.
The authors are thankful to the Department of Cardiovascular and Thoracic Surgery, King George's Medical University, Lucknow.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]