Mediated Modular Rna Guided Regulation Of Transcription Essay
1771 Words Nov 28th, 2015 null Page
Overview of CRISPR and this paper
The CRISPR technology is a genome editing technology which uses RNA-guided endonucleases from the prokaryotic adaptive immune system. CRISPR stands for clustered regularly interspaced short palindromic repeats and refers to DNA loci. Cas9, an endonuclease, is targeted by a short guide RNA, which pairs with the target DNA. In the natural mechanism, the target DNA represents the phage sequence, but this can be replaced by the sequence of interest in genome engineering to retarget Cas9.
In addition to genome editing, the CRISPR module can also be used for RNA-guided regulation of transcription of eukaryotes. A catalytically inactive Cas9 protein (dCas9) can bind to a transcriptional repression to silence expression or can bind to an effector to activate a gene. This process is known as CRISPR interference or CRISPRi. To improve the efficiency of repression, the dCas9 gene was fused with repressive chromatin modifier domains such as the KRAB (Krüppel-associated box) (Gilbert 443). The resulting gene expression showed a 15-fold repression for the best sgRNA as compared to the 2-fold repression using only dCas9.
This method showed minimal off-target effects, and was also shown to knock down endogenous expression of select genes (CD71 and CCR4). The repression efficiency of dCas9 depended on the affinity of the KRAB domain to certain regions of the DNA. Some genes…