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Transcription Deactivation using dCas9

dCas9, also referred to as endonuclease deficient Cas9 can be utilized to edit gene expression when applied to the transcription binding site of the desired section of a gene. The optimal function of dCas9 is attributed to its mode of action. Gene expression is inhibited when nucleotides are no longer added to the RNA chain and therefore terminating elongation of that chain, and as a result affects the transcription process. This process occurs when dCas9 is mass-produced so it is able to affect the most amount of genes at any given time via a sequence specific guide RNA molecule. Since dCas9 appears to down regulate gene expression, this action is amplified even more when it is used in conjunction with repressive chromatin modifier domains. The dCas9 protein has other functions outside of the regulation of gene expression. A promoter can be added to the dCas9 protein which allows them to work synergistically with each other to become efficient at beginning or stopping transcription at different sequences along a strand of DNA These two proteins are specific in where they act on a gene. This is prevalent in certain types of prokaryotes when a promoter and dCas9 align themselves together to impede the ability of elongation of polymer of nucleotides coming together to form a transcripted piece of DNA. Without the promoter, the dCas9 protein does not have the same effect by itself or with a gene body.

When examining the effects of repression of transcription further, H3K27, an amino acid component of a histone, becomes methylated through the interaction of dCas9 and a peptide called FOG1. Essentially, this interaction causes gene repression on the C + N terminal section of the amino acid complex at the specific junction of the gene, and as a result, terminates transcription.

dCas9 also proves to be efficient when it comes to altering certain proteins that can create diseases. When the dCas9 attaches to a form of RNA called guide-RNA, it prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism's genome. Essentially, when multiple repeat codons are produced, it elicits a response, or recruits an abundance of dCas9 to combat the overproduction of those codons and results in the shut-down of transcription. dCas9 works synergistically with gRNA and directly affects the DNA polymerase II from continuing transcription.

Further explanation of how the dCas9 protein works can be found in their utilization of plant genomes by the regulation of gene production in plants to either increase or decrease certain characteristics. The CRISPR-CAS9 system has the ability to either upregulate or downregulate genes. The dCas9 proteins are a component of the CRISPR-CAS9 system and these proteins can repress certain areas of a plant gene. This happens when dCAS9 binds to repressor domains, and in the case of the plants, deactivation of a regulatory gene such as AtCSTF64, does occur.

Bacteria are another focus of the usage of dCas9 proteins as well. Since eukaryotes have a larger DNA makeup and genome; the much smaller bacteria are easy to manipulate. As a result, eukaryotes use dCas9 to inhibit RNA polymerase from continuing the process of transcription of genetic material.

CITATIONS:

Jensen et al. Microb Cell Fact (2017) Transcriptional reprogramming in yeast

using dCas9 and combinatorial gRNA strategies 16:46

DOI 10.1186/s12934-017-0664-2

O’Green Henriette et al. dCas9-based epigenome editing suggests acquisition

of histone methylation is not sufficient for target gene

Repression Nucleic Acids Research, 2017, Vol. 45, No. 17 9901–9916

Pinto, S. Belinda et al. Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9 Molecular Cell 68, 479-490 November 2, 2017, 2017 Elsevier Inc.

'Lowder L.G., Paul J.W., Qi Y. (2017) Multiplexed Transcriptional Activation or Repression in Plants Using CRISPR-dCas9-Based Systems. In: Kaufmann K., Mueller-Roeber B. (eds) Plant Gene Regulatory Networks. Methods in Molecular Biology, vol 1629. Humana Press, New York, NY (website specifically asked this reference to be cited as seen above)'

'Barrangou, R. & Horvath, P. A decade of discovery: CRISPR functions and applications. Nat. Microbiol. 2, 17092 (2017). (article specificially wants reference to cited as seen above)'