CRISPR-based Epigenetic editing (Part 53- CRISPR in Gene Editing and Beyond)
Welcome to the 53rd part of the multi-part series on applications of CRISPR in gene editing and beyond.
The second level of genomic regulation after transcription effectors is epigenetic gene regulation. This process involves a series of chemical modifications that results in selective silencing or activation of particular genes without direct altering the DNA primary sequence. Epigenetic modifications can occur within the DNA itself or on the histone proteins that assist in DNA packaging. These modifications play a crucial role in deciding whether genes are activated or suppressed, thereby influencing protein production within cells. This regulatory mechanism helps guarantee that each cell produces solely the proteins that are necessary for its proper function.
A common type of epigenetic modification is DNA methylation. DNA methylation involves the attachment of methyl groups to the C5 position of cytosine residues in CpG dinucleotide sequences of the DNA. The presence of methyl groups in the promoter blocks the binding of transcription factors, and RNA polymerase II; thus, the transcription of the gene is inhibited, causing the gene to be turned off or silenced, and therefore, no protein is produced from that gene.
Histone modification is another prevalent form of epigenetic modification. Histones are structural proteins that aid in the packaging of DNA within the cell nucleus. They can be modified by the addition or removal of chemical groups like methyl or acetyl groups. These chemical groups impact how tightly DNA is wrapped around the histones and whether a given genomic sequence is stretched out and accessible to the transcriptional machinery, affecting a gene to be turned on or off. For instance, the addition of acetyl groups to the lysine residues of histone proteins causes histones to drift away from DNA. This released structure facilitates the binding of transcription factors and RNA polymerase II to the target gene, thus activating its transcription. On the other hand, histone deacetylation, i.e., the removal of acetyl groups, blocks access to transcriptional machinery, thus, repressing the transcription of the target gene.
Also, the addition of methyl groups to the histone proteins can either activate or repress the transcription of the gene, thus turning it on or off. For example, trimethylation of histone H3 at lysine 4 (H3K4me3) in the promoter region activates the transcription of the gene. However, dimethylation of histone H3 at lysine 9 (H3K9me2), is a signal for transcriptional silencing.
Thus, the epigenome, which consists of chemical compounds that modify genomic DNA and histones, acts as the second level of gene expression regulation after the transcription effectors.
To achieve CRISPR-Cas9 mediated epigenetic regulation, dCas9 can be fused to various effectors.
We have already discussed in Part 30 that dead Cas9 or dCas9 is a mutant in which both cleavage domains HNH and RuVC of Cas9 are inactivated. Although dCas9 can no longer cleave DNA, it can still bind target DNA with the same precision when guided by sgRNA.
One such effector is histone acetyltransferase p300, which can be fused to dCas9, creating a dCas9-p300 fusion activator (Hilton et al., 2015; Chen et al., 2017; Kuscu et al., 2019). p300 adds acetyl groups to the histone proteins and facilitates the binding of transcription factors and RNA polymerase II to the target gene, thus significantly activating its transcription. Furthermore, the dCas9-p300 fusion achieves higher activation than dCas9-VP64 alone (discussed in Part 51).
Another transcriptional activator is Tet1 demethylase, which, when fused to dCas9, decreases methylation and induces transcription at CpG regions of the promoter of the target gene (Gjaltema and Rots, 2020).
In contrast to transcriptional activators, transcriptional repressors DNA Methyltransferase 3 Alpha, abbreviated as DNMT3A, and Lysine-specific histone demethylase 1 abbreviated as LSD1, repress transcription of the target gene. DNA Methyltransferase 3 Alpha, when fused to dCas9, causes cytosine methylation of the promoter of the target gene, thus repressing its activation. On the other hand, LSD1 is a histone demethylase that, when fused to dCas9, targets histone H3, methylated at lysine 4 residue, and demethylates it, leading to transcriptional repression of the target gene.
This is how dCas9 can regulate the target gene expression.
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