could the crispr/cas9 system be applied to atherosclerosis? · (2015) show us that a unique...
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Journal of Cardiology & Clinical Research
Cite this article: Olivaes J, Markoski M (2016) Could the CRISPR/Cas9 System be applied to Atherosclerosis? J Cardiol Clin Res 4(3): 1064.
*Corresponding author
Markoski, M. Laboratório de Cardiologia Molecular e Celular, ICFUC, Av Princesa Isabel, 370, Porto Alegre, RS, Brazil, CEP 90620-001, Tel: 5551-32232746;
Submitted: 15 March 2016
Accepted: 13 April 2016
Published: 15 April 2016
Copyright© 2016 Markoski et al.
OPEN ACCESS
Keywords•CRISPR/Cas9•Genome editing•Mutations in atherosclerosis•Molecular approaches for coronary artery disease
Mini Review
Could the CRISPR/Cas9 System be Applied to Atherosclerosis?Jessica Olivaes1,2 and Melissa Markoski1*1Department of Molecular and Cellular Cardiology Laboratory, University Foundation of Cardiology, Brazil 2Department of Molecular and Cellular Cardiology Laboratory, Federal University of Health Sciences of Porto Alegre, Brazil
Abstract
The genome editing is a tool continuously exploited in the search for repairing mutations that cause diseases. Recently, the CRISPR/Cas9 system, a defense mechanism found in bacteria, has been receiving special attention due to the ease of replacing erroneous bases to DNA sequence through specific nucleases. CRISPR/Cas9 technology is cheap, specially compared to gene therapy, and has been studied as a possibility in curing genetic disorders, acting only in mutant alleles, in several murine models. Recently, the FDA approved the use of a genomic editing tool, similar to the CRISPR/Cas9 system, for production of factor IX in patients with severe hemophilia B, a monogenic disorder. However, most cardiovascular diseases are multi factorial, specially coronary artery disease, and are caused by mutations not only in genes, but also in regulatory proteins key to the functions of these genes. Here, we reviewed the applications of CRISPR/Cas9 methodology in the cardiovascular area and speculate its possible application to atherosclerosis, highlighting features and potential risks.
ABBREVIATIONSCAD: Coronary Artery Disease; CRISPR: Clustered Regularly
Interspaced Short Palindromic Repeats; Cas9: CRISPR associated protein 9; SNPs: Single Nucleotide Polymorphisms
INTRODUCTIONAmong the cardiovascular diseases, coronary artery disease
(CAD) singly kills more people or brings serious consequences in morbidity, leading to economic problems and raising expenditure in public health [1], both in industrialized and in “in development” countries [2].
By its pathological and physiological circumstances, CAD, stimulated by ischemic events and risk factors, leads to the development of plaque, which can have more serious consequences, such as endothelial dysfunction, myocardial infarction and, ultimately, risk of death [3]. It is well known that optimized pharmacological treatments, surgical interventions, for example, angioplasty and coronary artery bypass graft, as well as innovative attempts such as cell therapy, are unable to cure the disease; they only minimize some of the clinical conditions in non-refractory patients [4,5]. This situation deems as imperative the search for strategies that, in combination with treatment procedures, may lead to the reversal of the disease, at least in part.
Recently, approaches that allow the editing of genomes have
emerged. An example is the Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR)/CRISPR associated protein 9 (Cas9) system (Figure 1). In short, the system consists of an immune response mechanism identified in the bacterium Streptococcus pyogenes, which is directed to phages and plasmids, where, after contact with the invader, small fragments of RNA are transcribed from the palindromic repeats that recognize the specific exogenous DNA, acting as a guide to nuclease cleavage and consequent elimination of this exogenous DNA [6] (Figure 1A). Thus, the in vitro system may be extended to the genes of any other cell, once the RNA sequences may be synthesized in the laboratory. This allows the excision of sequences “damaged”, which, in turn, activates the cellular mechanisms of repair and “error correction” [7] (Figure 1B). Thereby, the most common use of the CRISPR system is for repairing (editing) sequences. Besides, the technique may also be used to decrease or suppress the expression of defective genes. However, a question remains: could the use of this easy editing system, whose application has been proposed in several diseases (with tests in vitro and in several animal models), be effectively expanded in order to respond to a disease as complex as CAD? If we consider that small mutations can do great damage, the answer is that minor repairs might generate great changes. Accordingly, we must turn our attention to the single nucleotide polymorphisms (SNPs) present in genes that singly contribute to the development of CAD.
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Figure 1 The CRISPR/Cas9 system. (A) The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR Associated (Cas) system was discovered in bacteria as a defense mechanism against foreign DNA, for instance bacteriophage but also virus and plasmids. Once the invader injects its genetic material in the bacterium, the insertion may occur in form of spacers into the chromosomal double-strand DNA in a region of small repetitions, and identified along with an adjacent protospacer motif sequence (PAM). There are a set of genes for enzymes with nuclease (Cas9) and helicase (Cas1 and Cas2) functions nearest the CRISPR sequences. Apart from these, there is a sequence named trans-activating (Trac)-CRISPR RNA (crRNA) that plays a role in the maturation of crRNA. The Trac-crRNA is complementary (in some base pairs) with the pre-crRNA, forming a RNA duplex. This is cleaved by RNase III (processing), and constitutes a mature crRNA/Trac-crRNA hybrid. The hybrid acts as a guide RNA for the endonuclease Cas9, which after further invasion and following recognition causes breaks in double-stranded DNA, constituting an elegant acquired immune response. (B) Reconciling sophisticated molecular and biotechnology techniques, the CRISPR/Cas9 system was engineering for application in genome editing, being commercially available for thousands of targets. The guide RNA can be manufactured in vitro against any genomic target site. Both guide RNA and Cas9 can be delivered to the cells using different mechanisms, such as vectors or biochemical agents. The system is directed to the nucleus, where the recognition of the target sequence and the consequent DNA break occur. The natural cellular repair mechanisms are triggered, redoing the breaks, removing erroneous inserts in order to correct the gene function or promoting deletions introduced to inactivate harmful genes. Additionally, once CRISPR/Cas9 is proposed for the embryonic stage in animal models, the progeny might generate founders (by recombination) with knock out or knock in allele mutations, therefore activating or inactivating genes.
How to access specific polymorphisms for CAD
In genetic terms, CAD is a highly complex and heterogeneous disease. For several years, the disease is completely asymptomatic and may cause angina pectoris and sudden death. The Framingham Heart Study reported that one-third of heart attacks had unknown causes [8]. However, nowadays, molecular analysis tools are being increasingly optimized,
bringing important information to the Translational Medicine. As an example, it might be highlighted the following sequencing methods of genomes and/or gene expression products such as the whole-exome sequencing, which has been able to identify various factors linked to CAD. Thus, mutations, variants and dominance relations can be determined, evidencing the presence of specific SNPs in sequences involved with hypercholesterolemia,
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formation of plaque, activation of inflammatory process and cell recruitment, among other mechanisms [9,10]. Stitham et al. (2011) related 18 new non-synonymous mutations found in prostacyclin receptors, G protein-coupled, with decreased levels of cyclic AMP and compared with clinical data for the presence of coronary heart disease [11]. Genome-wide association studies have revealed many new mutations associated with CAD where, in many ways, the polymorphisms may be linked with plasma levels of target proteins. An example is the association between 2 recently identified polymorphisms (rs1746048 and rs501120) and levels of the CXCL12 chemokine and its biological relationship with atherosclerosis [12]. Rai et al. (2014), in a meta-analysis, also shows the association of polymorphisms present in the endothelial nitric oxide synthase gene and CAD, emphasizing the presence of specific SNPs with CAD’s risk in Middle Easterners and Asians [13]. Interestingly, today there are also public repositories where the SNPs are deposited and accessed, facilitating the search for specific mutations in target genes. Thus, the identification of mutations in exons (gene coding regions) and also in the gene regulatory regions (transcription promoters and cis elements) may facilitate the identification of targets. Importantly, these SNPs also allow the development of transgenic animals where, in fact, the mutation may be studied in vivo and subsequently compared to the clinical data.
Outlook in accessing CRISPR/Cas9 technology for CAD
Some considerations are important about choosing polymorphisms-targets for the use of CRISPR/Cas9 system. First, it must be taken into consideration the dominance relationship of the gene of choice, because this allows the desired biological response of the system. In this case, it is also important to know if the phenotype is expressed from one or multiple alleles (the latter is not desirable). In rats, the synthetic guide RNA (Figure 1B) leads to allele-specific editing of the dominant phenotype by an interallelic conversion between homologous chromosomes during cell division [14]. Second, the researcher must also predict whether it will edit only one or more bases in the same sequence, due to possible physical implications that might hamper the delivery of components of the system, in addition to increase the costs of purchase. Here, Ousterout et al. (2015) show us that a unique multiplex gene editing through CRISPR/Cas9 system enabled the generation of a single large deletion, which could correct up to 62% of dystrophin mutations in Duchene muscular dystrophy [15]. Additionally, there is also a legitimate concern with the off-target delivery [16]. A study with a primate model showed that the off-target effect is site-dependent and can be minimized by optimizing the procedure [17]. Further, a difficulty that can be pointed out now is that the technique, considering its molecular strategy, must be carried out on the premises of a molecular biology laboratory, by trained human resources and with clinical quality control, especially if it is being considered for use in patients.
Nonetheless, the CRISPR/Cas9 technology may be exploited for atherosclerotic diseases like CAD, even more when there is increasing accessibility to single polymorphisms involved with the disease. Yang et al. (2014) performed a study in rabbits, a model for atherosclerosis, projecting the guide RNA for targeting genes such as apolipoprotein E (ApoE), cluster of differentiation
36 (CD36), low-density lipoprotein receptor (LDLR), leptin, ryanodine receptor 2 (RyR2), leptin receptor, among others. The authors achieved success in all gene editing and, even more, the off-target effect has not been observed [18]. Similarly, it has been shown that the CRISPR/Cas9 system was efficiently capable in generating loss-of-function of the protein convertase subtilisin/kexin type 9 (PCSK9), which is in vivo related to lower levels of cholesterol [19]. Together, these results provide a light to translation for humans. Although its application in human cardiovascular disease might be a little far from the current reality, the tool, which may be easily obtained commercially, is already being applied in studies on some cases of cancer [20], another multi factorial and complex disease. However, it should be remembered that, like any new technology, the risks, safety, physiological adaptation, maintenance of homeostasis, implications at immune response and other questions still need to be thoroughly studied.
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Olivaes J, Markoski M (2016) Could the CRISPR/Cas9 System be applied to Atherosclerosis? J Cardiol Clin Res 4(3): 1064.
Cite this article
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