Chapter Thirteen - CRISPR/Cas9 in epigenetics studies of health and disease
Introduction
Epigenetics is known as the chemical modification of gene that affects the phenotypic changes without altering the gene sequences. The phenotypic changes are heritable, but, do not modify genetic information. In eukaryotes, genomic DNA is compactly packed into chromatin by forming nucleosomes. Each nucleosome comprises a histone octamer wrapped around by approximately 146 base pairs of DNA. A histone octamer is formed by two copies of each histones H2A, H2B, H3 and H4. The linker histone H1connects two adjacent nucleosomes. The extended histone tails from the nucleosome are covalently modified, include methylation, acetylation, phosphorylation, sumoylation, ubiquitination and ADP ribosylation, non-coding RNAs interactions.
Section snippets
Why epigenetic modification is required?
Covalent modifications of histones change chromatin structure. The change of structure is either open chromatin or “active” state and closed chromatin or inactive state. Activation of a gene requires an active or open chromatin state facilitate to the gain access of the transcription factors to the promoter DNA. Then the gene can be transcribed. Acetylation, phosphorylation or sometimes histones methylation is associated with an open state of chromatin cause gene activation. Deacetylation of
History of epigenetics
Epigenesis theory was developed by C. F. Wolff (1734–1794) a naturalist and physician from Germany. He had proposed that during the process of developing structures that arise (Wolff 1759, 1764). In the 1940s Conrad Waddington introduced the term epigenetics. The meaning of the word “epigenetic” is “in addition to changes in genetic sequence.” Epigenetic comprises a process that changes gene activity without altering the DNA sequence and causes modifications that can be inherited to daughter
Types of Epigenetic modification
Broadly, epigenetic modifications are of two types.
How the role of epigenetics is important about the different physiological process?
These post-transcriptional modifications found in histones of chromatin cause epigenetic modifications. They can interplay to modulate the chromatin structure, which acts on important molecular and cellular processes like DNA repair, transcription, cellular development, differentiation and homeostasis, etc.19, 20, 21 However, a variety of epigenetic changes cause diseases like cancer, diabetes, neurological disorder, infectious diseases, autoimmunity, etc.22, 23, 24, 25, 26 Furthermore, a
Clinical use of epigenetics studies
The two broad concerns in the clinical use of Epigenetics studies are i. identification of disease biomarkers and ii. development of their therapeutics.
CRISPR/Cas9
CRISPR (Clustered Regularly Interspaced Palindrome Repeats) associated protein 9 (Cas9) system, abbreviated as CRISPR/Cas9 is an RNA dependent nuclease. This system is found in bacteria to protect the bacterial cells from viral infection. CRISPR is the single guide RNA (sg RNA) (20 nt long) part having target sequence and RNA guided Cas9 endonuclease. In the presence of sg RNA, Cas 9 endonuclease comes in on to the target sequence and makes a double-stranded break to the upstream
Modulating cancer epigenetics
Earlier, we have discussed differential epigenetic patterns observed in different types of cancers.35, 36 Promoter Hypermethylation of tumor suppressor genes suppresses many tumor-suppressing functions. On the other hand, hypomethylation causes activation of the proto-oncogenes. Thus, epigenetic alterations are the basis for the instability of genome and cancer development.57, 58 Developed epigenetic therapeutics have been used to treat cancer88, 95 that generally change the epigenetic
Limitation
Even though the technique has vast therapeutic potential, it is essential to address different challenges before its use in the field of gene therapy and clinical applications. The specificity of the CRISPR/Cas9 technique is very essential for its use in clinical studies. Like epigenetic- drugs, CRISPR-Cas9 tools cause off-target gene editing.184 Therefore, different strategies are developed to check possible off-target CRISPR mutations, and new systems, protocols, are under trial to minimize
Conclusion and future perspectives
Despite many challenges, CRISPR/Cas9 mediated epigenome editing has been widely used in various fields due to its high efficiency, low cost, and flexibility.190, 191 Moreover, dCas9 tools are used for characterizing the new regulatory elements to investigate their biology and functions in the genome.192
Furthermore, epigenome editing has been used for modifying cell lines and organisms to investigate pathophysiological mechanisms behind various genetic diseases. This will enable the necessary
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