Download Gilbert - Blumberg Lab

Genome-Scale CRISPR-Mediated Control of
Gene Repression and Activation
By Luke A. Gilbert et. al.
Presented by: Sabrina Will and Ashley Boydd
Background
-At the time of this paper, permanently modifying or deleting DNA could
be done with customized zinc finger proteins, TALE (transcription
activator-like effector) endonucleases, and CRISPR/Cas9
-However, zinc finger and TALE require unique fusion proteins in order
to modulate transcription rather than simply knock out a gene, which
would be rediculous to try and use for genome-wide screens
-Programmed RNAi molecules could be used to knockdown mRNAs,
but they had too many off-target effects
What is CRISPR?
• First Discovered in archaea and later in
bacteria; serves as a primitive immune
system against viruses
• “spacer” sequences are remnants of
genetic code from invaders
• If spacers are complimentary to a
sequence, degradation of the nucleic acid
occurs
• There are a few types of CRISPR systems
but type II (CRISPR-Cas9) is most
commonly used
Important Components of CRISPR
Two main components:
• gRNA (guide RNA): contains
sequence to be targeted and
necessary for cas9 binding
• Cas9: acts as an endonuclease
Limitations of CRISPR-CAS9
-Causes irreversible frameshift disruptions, meant for knocking
out genes, which makes it difficult to study essential genes and
lnRNAs
- Double stranded breaks can be cytotoxic
-Cells can be good at accidentally fixing the damage due to errorprone DNA repair, limiting the ability to knock out all alleles
Gilbert’s Previous Study
-The year before this paper was published, Gilbert and his team developed a
modified CRISPR/Cas9 technology called CRISPRi, which was able to
repress genes at the transcriptional level
-Worked one of two ways:
-1. Directly blocking RNA polymerase activity (dCAS9)
-2. Through effector domain-mediated transcriptional silencing (dCAS9KRAB)
- They next wanted to better understand and optimize this new tool they had
developed
Primary Aims
• Develop and test a method for high-specificity, genomescale modulation of transcription of endogenous genes in
human cells using CRISPRi/a
• Aimed to accomplish this in three steps:
1. Perform a saturating screen to test the activity of every unique sgRNA
broadly tiling around transcription start sites of 49 genes known to
modulate cellular susceptibility to ricin
2. From the screen, extract distinct rules for regions where CRISPRi/a
maximally changes the expression of endogenous genes and also for
predicting off-target effects
3. Validate these libraries by screening for genes that control cell growth
and response to a chimeric cholera/diphtheria toxin
Step 1: Saturating screen
• Used massively parallel oligonucleotide synthesis to generate a library
of sgRNAs that tile a 10kbp window around the TSS of 49 genes known
to contribute to ricin resistance
• 54,810 total sgRNAS
• The entire library was transduced into K562 human myeloid leukemia
cells stably expressing dCas9 or dCas9-KRAB using lentiviral particles
• Then used deep sequencing using the sgRNAs as barcodes to count
the frequency of each sgRNA after growing cells in either standard
conditions or with exposure to ricin
CRISPRi Tiling Screen
• Define a set of rules for construction of a genome scale CRISPRi library
• Use ricin resistance phenotype to measure transcriptional repression
CRISPRi Tiling Screen (cont.)
• Compare the phenotype
strength of CRISPRi with
previously published
shRNA data
• CRISPRi shows much
stronger phenotype in
most cases than shRNA
CRISPRi Tiling Screen (cont.)
• Took 30 sgRNAs and created
mismatch base pairing
• CRISPRi activity dramatically
decreased with just a single
mismatch
• Shows CRISPRi results in
high specificity with very little
off target repression activity
CRISPRa Tiling Screen
• Developed new CRISPRa
method termed sunCas9
• One sgRNA with one binding site
is sufficient to activate
transcription
• dCas9 fusion protein bound to
DNA recruit multiple copy of
activating effector domain
• What are the optimal conditions?
CRISPRa Tiling Screen (cont.)
• Ricin resistance phenotype peaks downward closest to the TSS,
demonstrate CRISPRa actually works!
• Implies similar to CRISPRi, CRISPRa works best near the start of TSS
Step 2: Extracting Rules for optimal use of
CRISPRi/a for genome-wide use
From CRISPRi/a tiling screens, were able to extract rules for optimal use of
both systems:
• CRISPRa works best −400 to −50 bp upstream from the TSS while
CRISPRi works best −50 to +300 bp relative to the TSS of a gene
• CRISPRi/a are both extremely specific systems that result in very little off
targeting effects
Step 3: Validating Libraries by Screening
• Gilbert and his team demonstrated the application of their
CRISPRi/a technology in two ways:
1. Screening for genes that control cell growth
2. Screening for genes that govern a response to a CholeraDiphtheria fusion toxin (CTx-DTA)
Screening for genes that control cell growth
• First screened for genes essential in
cell growth of K562 cells
• Showed that no off target effects were
happening in K562 cells; Y
chromosome and Olfactory genes
unaffected
• Demonstrated no toxicity occurring
under these conditions
Screening for genes that control cell growth (cont.)
• Synthesized and Cloned genome-scale
CRISPRi sgRNA library targeting 15,977
human protein coding genes
• Used metric of average growth phenotype
to identify hit genes
•
Validating Gilbert’s approach as a
screening platform.
Revealing Response Pathways for a Cholera-Diphtheria Fusion
Toxin from a CRISPRi/a Screen
A. Model for CTx-DTA binding, retrograde trafficking,
retrotranslocation, and cellular toxicity
B. Overview of top hit genes detected by CTx-DTA screen.
Dark red/blue circles represent top 50 sensitizing and
protective hits, light red/blue circles represent other
proteins that fall into the complexes
1. Red= causes sensitivity to toxin, Blue= reduces
sensitivity to toxin, Stars= previously identified by
haploid mutagenesis screen
2. Circle area is proportional to phenotype strength
C. CRISPRi/a hits in sphingolipid metabolism, which is
responsible for toxin uptake
Overall Conclusions
• CRISPRi/a results are highly reproducible
• Any intrinsic toxicity is undetectable
• Transcriptional repression using CRISPRi is inducible,
reversible, and can target essential genes
• CRISPRi/a can be used to control transcript levels for
endogenous genes across a broad range
• Properly designed CRISPRi reagents are highly specific
Criticism
• Collections of figures were disjointed and out of order; should
have dispersed figures throughout text as opposed to lumping
them all into a page
• Lots of information; felt that some experiments should have
had their own papers
Future Readings
• CRISPR/Cas9 and Targeted Genome Editing: A New Era in
Molecular Biology by New England BioLabs
• CRISPR-mediated modular RNA-guided regulation of transcription
in eukaryotes. Cell. 2013;154:442–451 by Gilbert et al.
References
• Gilbert et al. 2014. Genome-Scale CRISPR-Mediated Control
of Gene Repression and Activation. Cell; 159:647-661.
• Gilbert et al. 2013. CRISPR-Mediated modular RNA-guided
regulation of transcription in eukaryotes. Cell; 154, 442-451.