genome-scale crispr-mediated control of the gene repression and activation luke a. gilbert, max a....

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

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

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

CRISPR Cas9 Introduction

Experiment

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

Ricin-resistance phenotypes, comparing CRISPRi and

sgRNAs for genes previously established to cause ricin-

resistance phenotypeswhen knocked down by RNAi.

Green line: median sgRNA activity in a

defined window for all genes.

Orange region:observed average window of maximum CRISPRi activity

dCas9-SunTag sgRNA system for CRISPRa

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

CRISPRi knockdown and CRISPRa activation of the

same gene can have opposing effects on ricin

resistance

Coexpression of sgRNAs and dCas9-KRAB or dCas9-SunTag is not toxic in K562 cell lines

over 16 days

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

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