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CRISPR Cas 9: Beginning of a new era of cardiovascular genomics
The growing appreciation of human genetics and genomics in cardiovascular disease (CVD) accompanied by the technological breakthroughs in genome editing, particularly the CRISPR-Cas9 technologies, has presented an unprecedented opportunity to explore the application of genome editing in cardiovascular medicine. In the latest issue of Trends in Cardiovascular Medicine, Zhang et al have highlighted the recent advances of the CRISPR-based genome editing toolbox and discussed the potential and challenges of CRISPR-based technologies for translating GWAS findings into genomic medicines.
The History of gene editing:
In 2005, Mojica et al. reported on the ability of CRISPR to prevent bacteriophage infection in bacteria carrying the specific bacteriophage DNA sequences in the spacer between the CRISPR elements. CRISPRs (clustered regularly interspaced short palindromic repeats) consist of repetitive DNA sequences 25–50 nucleotides in length that are interspersed with non-repetitive elements (spacers) of similar length. The second piece of the gene editing puzzle was provided later that same year by Bolotin et al., who described Cas9, a CRISPRassociated protein with nuclease activity, allowing CRISPR/Cas to degrade the DNA of the invading bacteriophage.
The advent and continued advancement of CRISPR-Cas9 technologies enable manipulations of the human genome with increasing precision and efficiency.
CRISPR-Cas9 based genome editing technologies:
The three related technologies in the CRISPR-Cas9-based toolbox (Figure 1) are:
1. Conventional CRISPRCas9. Cas9 nuclease creates DSB at location determined by gDNA and PAM. NHEJ generates random indels, often leading to premature stop codons and gene knockout. HDR allows desired mutagenesis according to donor template.
2. Base editors. dCas9 is fused to cytidine deaminase (cytidine base editors) or adenosine deaminase (adenine base editors). Base editors introduce point mutations by deamination without creating DSBs. Cytidine base editor is shown as an example.
3. Prime editing. dCas9 is fused to an engineered reverse transcriptase (RT). Prime editing gRNA includes an RT template, allowing direct rewriting of DNA.
The major limitations of CRISPR:
1. Off-target mutagenesis remains a major problem as the lack of precision raises safety concerns for clinical applications. High-fidelity Cas9 variants have also been developed to reduce non-specific DNA contacts and reduce off-target events.
2. Limited targeting capacity is another drawback. Because PAM is required for Cas9 recognition and binding, a DNA sequence is targetable only if a PAM sequence is present adjacent to it.
How well this technology fits the arena of cardiovascular medicine?
Several genetic loci have been implicated in monogenic inheritance of CVDs, including MYH7 in familial hypertrophic cardiomyopathy, H-RAS and HERG in long QT syndrome, and LDLR and PCSK9 in familial hypercholesterolemia. However, most common CVDs, such as coronary artery disease and myocardial infarction, are complex diseases that are driven by multiple variants of small to moderate effects.
Over the past decade, the advent of large-scale genotyping technologies, genome-wide association studies (GWAS), and more recently, phenome-wide association studies (PheWAS), has provided much more powerful tools to identify genotype-phenotype associations. GWAS detects associations between a variety of genetic variants with a particular physiological or clinical phenotype; PheWAS explores the relationship between a wide range of phenotypes and a specific genetic variant.
CRISPR-Cas9-based genome editing has opened a range of new opportunities to easily introduce and systematically characterize the effects of genetic variants identified in GWAS with cellular and animal models.
Translating CRISPR-Cas9 modalities into genomic medicines:
In the realm of cardiovascular medicine, there have not yet been any human trials exploring the use of CRISPR-Cas9 genome editing for therapeutic purposes. However, the application of genome editing tools for the prevention of complex CVDs may be achievable in the clinic in a nearer future.
GWAS has identified a number of naturally occurring pLoF mutations associated with reduced plasma lipid levels and lower risks of CVD including those found in PCSK9 and ANGPTL3. As carriers of these genetic variants are healthy individuals with reduced CVD risk, these naturally occurring pLoF mutations represent putative genetic targets for long-term protection against complex CVDs. Although CRISPRCas9-mediated genome editing for CVD prevention remains mostly theoretical, pre-clinical models have shown promising results.
Delivery options for CRISPR based therapy have been classified into three groups: physical delivery, viral vectors, and non-viral vectors. Viral vectors remain the most common CRISPR-Cas9 delivery vectors.
Donahue notes in an accompanying editorial that "an additional issue that should have been no surprise given the bacterial source of the CRISPR/Cas proteins is the induction of mammalian immune responses to the gene editing machinery." However he adds, "With patience and careful development, Crispr/Cas9 really does have the potential to change the way we practice medicine, in a good way."
Source: Trends in Cardiovascular Medicine
1. https://doi.org/10.1016/j.tcm.2020.06.008
2. https://doi.org/10.1016/j.tcm.2020.08.006
MBBS, MD , DM Cardiology
Dr Abhimanyu Uppal completed his M. B. B. S and M. D. in internal medicine from the SMS Medical College in Jaipur. He got selected for D. M. Cardiology course in the prestigious G. B. Pant Institute, New Delhi in 2017. After completing his D. M. Degree he continues to work as Post DM senior resident in G. B. pant hospital. He is actively involved in various research activities of the department and has assisted and performed a multitude of cardiac procedures under the guidance of esteemed faculty of this Institute. He can be contacted at editorial@medicaldialogues.in.
Dr Kamal Kant Kohli-MBBS, DTCD- a chest specialist with more than 30 years of practice and a flair for writing clinical articles, Dr Kamal Kant Kohli joined Medical Dialogues as a Chief Editor of Medical News. Besides writing articles, as an editor, he proofreads and verifies all the medical content published on Medical Dialogues including those coming from journals, studies,medical conferences,guidelines etc. Email: drkohli@medicaldialogues.in. Contact no. 011-43720751