genome-scale crispr-mediated control of the gene repression and activation luke a. gilbert, max a....
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![Page 1: Genome-Scale CRISPR-Mediated Control of the Gene Repression and Activation Luke A. Gilbert, Max A. Horlbeck, Britt Adamson, Jacqueline E. Villalta, Yuwen](https://reader034.vdocuments.site/reader034/viewer/2022052312/56649d6d5503460f94a4e644/html5/thumbnails/1.jpg)
Genome-Scale CRISPR-Mediated Control of the Gene Repression
and ActivationLuke A. Gilbert, Max A. Horlbeck, Britt Adamson, Jacqueline E. Villalta, Yuwen Chen, Evan H.
Whitehead, Carla Guimaraes, Barbara Panning, Hidde L. Ploegh, Michael C. Bassik, Lei S. Qi,
Martin Kampmann, Jonathan S. Weissman
By: Navjot Naur & Yazmin Rodriguez
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OverviewAlter transcription of endogenous genes using CRISPRi/a
o tested activity of sgRNA around transcription start site of
genes known to initiate cellular response to ricin (toxic protein)
extracted regions where CRISPRi or CRISPRa changed the expression of genes
algorithm to design two genomic libraries testing genes with sgRNA
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Background CRISPR Cas9
● Defense mechanism used in bacteria
● Has two components: guide RNA and Cas9 endonuclease
● Guide RNA consists of CRISPR RNA and tracr RNA
● can be used to cut any DNA sequence at a precise location
pnabio.com
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CRISPR Cas9 Introduction
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Experiment
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CRISPRi / CRISPRa
kampmannlab.ucsf.edu
• dcas9: catalytically dead version of Cas9
• dCas9-KRAB: catalytically dead version of Cas9 fused to a transcriptional silencer (KRAB domain)
• dCas9-SunTag: recruit transcriptional activators
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Ricin-resistance phenotypes, comparing CRISPRi and
sgRNAs for genes previously established to cause ricin-
resistance phenotypeswhen knocked down by RNAi.
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Green line: median sgRNA activity in a
defined window for all genes.
Orange region:observed average window of maximum CRISPRi activity
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dCas9-SunTag sgRNA system for CRISPRa
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Top:sgRNAs targeting VPS54
Green line: median sgRNA activity in a
defined window for all genes.
Orange region:observed average window of maximum CRISPRa activity
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CRISPRi knockdown and CRISPRa activation of the
same gene can have opposing effects on ricin
resistance
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Coexpression of sgRNAs and dCas9-KRAB or dCas9-SunTag is not toxic in K562 cell lines
over 16 days
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What they found● Control of transcript levels for endogenous genes across a high
dynamic range (up to ~1000-fold) reveals how gene dose controls function
● Mapping of complex pathways through complementary information provided by CRISPRi and CRISPRa
● CRISPRi provides strong (typically 90%–99%) knockdown of both protein coding and non-protein coding transcripts with minimal off-target activity
● CRISPRi is inducible and reversible, and can allow for the study of essential gene functions
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Application● probe the biological roles of all genes within the genome in a
single experiment
● Reveal mechanisms by which cancer cells develop resistance to anti-cancer drugs
● Identify cellular targets of new drugs
● Identify tumor suppressor genes that inhibit the growth of cancer cells
● Identify genes that regulate tissue development