Transcript Genome-Scale CRISPR-Mediated Control of the Gene

Genome-Scale CRISPR-Mediated
Control of the Gene Repression
and Activation
Luke 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
Overview
Alter 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 ricinresistance phenotypes
when 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 dCas9KRAB 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 offtarget 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