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PHARMACEUTICAL DRUG AND DESIGN, Genome-Wide RNAi screens, CRISPR-Cas9 -…
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Genome-Wide RNAi screens
Process by which RNA in dsRNA form, breaks down the mRNA for a specific gene, thus causing the silencing of the gene and also it stops the production of that protein
Initiation : generation of mature siRNA or miRNA from dsRNA
Execution : silencing of target gene/degradation or inhibition of translation
Applications
- Therapy
- Genome-Wide RNAi screens
- System Biology
Synthetic siRNA
can be applied to any organisms and are useful to carry out the GWRNA screenings ➔ candidate list ranked according to the z-scores reflecting the strengths of the phenotypes and these candidates are then going to be validated
RNAi vs Small molecules
RNAi: reduces the levels of target proteins
Small molecules: directly interfering with the activity
⤳ Aurora B
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CRISPR-Cas9
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rather than building RNAi libraries we can make CRISPR-Cas9 libraries which can be used with the goal of screen knock-outs
Initial 3 step process that bring to the formation of the Cas9-crRNA-tracrRNA complex that is ready to bind to invading DNA that is complementary to crRNA
a) DNA nicking : only one strand of the target DNA is cleaved by the HNH nuclease domain (is a domain of Cas9)
b) DNA double nicking : the DNA is cleaved in two sites to increase the genome-editing specificity using two different sgRNAs followed by repair though NHEJ
c) CRISPRi : transcription is blocked by targeting promoter regions with an endonuclease-inactive Cas9 variant
d) Transcription regulation : regulation of gene expression can be done by fusing the effector protein domain to Cas9 and target promoter regions