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Resources and applications of
TRC RNAi reagents in
National RNAi Core Facility
Lecturer: 林志隆 ( IMB&RNAi Core)
04/13/2010
The Nobel Prize in physiology
/medicine 2006
Goes to
Professor Andrew Z Fire, Ph.D.
Stanford University School of Medicine
Stanford, CA. USA
Professor Craig C. Mello, Ph.D.
University of Massachusetts Medical School
Worcester, MA, USA
RNAi: A gene silencing by dsRNA
RNA interference (RNAi)
A form of post-transcriptional gene silencing,
mimicking the effect of loss-of-gene-function.
RNAi does not result in stable genetic changes;
but in lower animal or plants, RNAi effects can be
inherited for one or two generations.
Timeline of RNAi achievements
Adapted from: http://www.invitrogen.com/etc/medialib/en/images/mainbody/
Thing/Data/Diagram.Par.12505.Image.-1.0.1.gif
Dr. R Jorgensen’s Experiment
C Napoli, C Lemieux, and R Jorgensen
Plant Cell. 1990 April; 2(4): 279.
• Attempts to overexpress
chalcone synthase by inserting
multiple copies of that gene into
the plant’s genome.
• Purple plants should become
purpler...
• Co-suppression: both
endogenous and introduced
genes silenced.
• PTGS – but what is the causative
factor?
PTGS= Post-Transcriptional Gene Silencing
PTGS in plants is due to small dsRNA
dsRNA hypothesis explained this phenomenon
Andrew J. Hamilton and David C. Baulcombe Science 1999 286: 950-52
Nature 391, 806-811 (19 February 1998)
Long dsRNAs trigger non-specific
silencing in mammalian
dsRNA
 C. elegans
 Drosophila
In mammal?
Long dsRNA initiate IFN response in mammalian
---global gene silencing
dsRNA-induced translation inhibition
in mammalian
Cytokine Growth Factor Rev. 2007 18:363-71.
How to Apply RNAi to Mammalian System
?
Effector of RNAi
長雙股RNA (long dsRNA)
cytosol
Gregory Hannon identified the “Dicer” –
an enzyme that chops double-stranded
RNA into little pieces.
small-interfering RNA; siRNA
-
Length of siRNA: 21 nts to 23 nts.
Nature. 2000;404:293-6; Nature. 2001;409:363-6
Nature 411, 494-498 (24 May 2001)
What Does siRNA Do
RISC: RNAi-induced silencing complex
Guide/ antisense strand
http://www.nature.com/focus/rnai/animations/index.html
http://www.pbs.org/wgbh/nova/sciencenow/3210/02.html
Mechanism of RNAi in mammalian cells
Antonin et. al ., 2007 Nature Reviews Drug Discovery 6, 443-453
Rational siRNA design
5’-P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
A
U
xG
A
3’’- OH
P- 5’’
3’’- OH
High thermal stability of the
5’ sense strand (SS) blocks
incorporation of SS into RISC
Low thermal stability of the 5’
anti-sense strand (AS) promotes
Incorporation of AS into RISC.
G or C is preferred.
AU rich is suggested.
Low stability in this region enhances RISC/AS-mediated
cleavage of mRNA and promote RISC complex release.
U at position 10 at SS is recommended.
Sense strand
Antisense strand
Reynolds et al. 2004
Other considerations
 no high GC content (35-65%);
 no inverted repeat sequence;
 no consecutive 3 Gs or 3 Cs;
 no consecutive 4 Ts if use polIII promoter;
Rational siRNA design
Crite
ria
Description
Score
1
Moderate to low (30%-52%) GC Content
Yes
1 point
2
At least 3 A/Us at positions 15-19 (sense)
3
Lack of internal repeats (Tm*<20¡ãC)
1 point
/per
A or U
1 point
4
A at position 19 (sense)
1 point
5
A at position 3 (sense)
1 point
6
U at position 10 (sense)
1 point
7
No G/C at position 19 (sense)
-1
point
8
No G at position 13 (sense)
-1
point
No
These characteristics are used by rational siRNA design algorithm to evaluate potential targeted
sequences and assign scores to them. Sequences with higher scores will have higher chance of success
in RNAi.
Reynolds et al. 2004
Phases of TRC Program
The RNAi Consortium (TRC)
The RNAi Core
Phase I (May/2004 to Apr/2007)
Jun/2005 to Apr/2008
Phase II (Oct/2007 to Sept/2011)
May/2008 to Apr/2011
Vector Used by RNAi Core
http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Functional_Genomics_and_RNAi/Product_Lines/shRNA_Library.html
Name
Description
U6 Promoter RNA generated with four uridine overhangs at each 3' end
Cppt/CTE
Central polypurine tract /constitutive transport element
hPGK
Human phosphoglycerate kinase eukaryotic promoter
puroR
Puromycin resistance gene for mammalian selection
SIN/LTR
3' self inactivating long terminal repeat
f1 ori
f1 origin of replication
ampR
Ampicillin resistance gene for bacterial selection
pUC ori
5' LTR
Psi
RRE
pUC origin of replication
5' long terminal repeat
RNA packaging signal
Rev response element
Configuration of TRC shRNA construct
Materials Received from TRC
 shRNA constructs and knockdown information:
TRC-I
TRC-II
Clone #
Gene #
Clone #
Gene #
Knockdown
Information
Human
83,117
16,026
53,070
10,149
41,774#1
Mouse
79,200
15,976
44,042
9,027
34,325#2
162,317
32,002
97,112
19,176
76,099
Total
#1
Targeting 7,074 genes
#2
Targeting 6,738 genes
 35 shRNA expression vectors (some are intermediates).
• with different selection/ fluorescence markers
04-11-2010 updated
TRC library performance
Good hairpins/gene coverage
Highest quality data: 10,711 genes
25%
16000
14000
20%
% genes
10000
8000
15%
10%
6000
5%
4000
2000
100-90
90-80
80-70
%KD
70-50
No KD
~0/5
~1/5
~2/5
~3/5
0
~4/5
0%
all HPS
#shRNA
12000
"good" clones/gene
TRC report
TRC validation (QRT-PCR)
Tests on 30,000 hairpin panels (~150,000 tests)
Released data on 14K distinct genes
35,000
Process
upgrade
successful
attempted
25,000
1,200 Genes / month
(6,000 shRNAs)
20,000
15,000
10,000
5,000
0
September
February
April
May
June
July
August
September
October
November
December
January
February
March
April
May
June
July
August
September
October
November
December
January
February
March
April
May
June
July
August
September
October
November
December
January
# genes (cumulative)
30,000
Resume
2006
2007
2008
2009
Throughput: 1,200  2,000 genes/month
2010
TRC report
Available Lentivirus
Item (arrayed, pooled, individual)
human kinase and phosphatase (hKP) subset
Mouse kinase subset / Mouse phosphatase subset
Human/ Mouse tumor suppressor subset
Human / Mouse transcription Factor subset
Human deubiqutinating enzymes subset
Control viruses
Pooled hKP
Pooled mouse kinase / Pooled mouse phosphatase
Pooled Human/ Mouse tumor suppressor set
Pooled Human / Mouse transcription Factor subset
Pooled Human deubiqutinating enzymes subset
Customized VSV-G pseudotyped lentivirus
80K Human shRNA Pool (16,026 genes)
Migration Profile of hTRC1
Pooled pDNA
Pool #
Ctl M
1
2
3
4
5
6
7
8
9 10 M
10K
6K
4K
3K
2K
86723
Clones
17745
Genes
80264
TRC1 Human Clones
16026
TRC1 Human Genes
4713
TRC1 Mouse Clones
1377
TRC1 Mouse Genes
1661
TRC2 New Human Clones
342
TRC2 New Human Genes
85
Control Clones
Expression of Hairpin RNA (shRNA)
Using Pol III Promoters
 Transcription initiation of DNA-dependent
RNApol III promoters (U6 or H1) are well
characterized. RNApol III transcription uses a
well-defined termination signal (TTTTT) and
the products have no extra sequence.
 Transcription from these promoters is very
efficient in various tissues.
Configuration/Structure of hU6 Promoter
-23
TATATAT
DSE
PSE
≈ 250bp
+1
-48
Structure of VSV-G-Pseudotyped
Lentivirus
Modified from http://www.washington.edu/alumni/columns/dec00/cells4.html
Replication of Retrovirus
http://www.accessexcellence.org/RC/VL/GG/retrovirus.html
Genome Organization of Lentiviral Vector
(Improved biosafety by eliminating non-essential genes or sequences)
pLKO.1-puro:
RSV promoter
Psi signal
R-U5
RRE
pbs
pCMVΔR8.91:
SD
Gag
Pol
hPGK promoter
AgeI
EcoRI
U3-R
U6 promoter
Puro
SA SD
SA
Rev
ΔΨ
VSV-G
pMD.G:
CMV promoter
SV40 PolyA
RRE
Tat
CMV promoter
PPT
SIN
SV40 PolyA
SV40 PolyA
HEK293T as Packaging Cells
Procedures:
Day1: seeding cells
Day2: co-transfection
Day3: re-fresh media
Day4: harvest viruses/
re-add media
Day5: harvest viruses
From genome sequence to gene function
Function Genomics
 What does the gene mean?
Forward and reverse genetics
approach to study gene function

Forward Genetics:
 start with a phenotype, find the gene.
 naturally occurring mutants can be used.

Reverse Genetics:
 start with a gene, determine its phenotype.
 identify the phenotypes resulting from the
disruption of a particular gene.
Applications of
RNAi libraries
RNA Interference
Cancer
•
•
•
•
•
Basic Research
Viral Infection
Gene functions
Tumor biology
Host factors required for viral replication
Biological pathways
And more
Approaches to large-scale RNAi
screen/selection
Arrayed RNAi library/Screen
siRNA Plasmid Vector Viral Vector
Pooled RNAi library
/Selection
Viral Vector
Transfection
Transduction
High Throughput Assay
for Altered Phenotype(s)
2nd assay to validate hits
Transduction
Selective screen for
Altered Phenotype(s)
Hits identification:
Barcode microarray/
RT-PCR sequencing
RNAi pooled screening: positive selection
R&D in RNAi
SAEC/Core
Control
Search for cellular factors that support primary human
small airway epithelial cell (SAEC) growth using RNAi
pooled selection, 17 genes that support SAEC growing in
soft agar are identified.
SAEC/ Control
SAEC/ RNAi
> 0.45 mm
SAEC/ RNAiAnchorage-dependent growth assay
RNAi pooled screening: negative selection
How to ensure that hits aren't off-target
◙ Off-Target:
◙ How/ Criterion:
 Phenotype change is caused by two or more independent
shRNAs that target the same gene
◙ Why:
Degradation of mRNA can occur by
two separate pathways in RNAi
Khvorova A. RNA (2008),14:853-861.
3’ UTR hexamer frequency in human genome
SCF: seed complementary frequency
high(>3800), medium (z2500–2800), or low (<350) SCFs
in the HeLa transcriptome
Khvorova A. RNA (2008),14:853-861.
Microarray signatures of GAPDH- and
PPIB-targeting siRNAs
One nt shift in seed
sequence:
GAPDH M1 sense: 5GGCUCACAACGG
GAAGCUU
GAPDH M8 sense: 5GCUCACAACGGG
AAGCUUG
Seed region not static
Same seed sequences in different target genes:
GAPDH H15 sense: 5-GAAGUAUGACAACAGCCUC
PPIB H17 sense:
5-CGACAGUCAAGACAGCCUG
Khvorova A. RNA (2008),14:853-861.
Seed sequence plays major role in off-target
Khvorova A. RNA (2008),14:853-861.
Configuration of TRC shRNA construct
shRNA processing
How are the TRC library shRNAs processed into short dsRNAs?
Implications: hairpin design, off-target effects
polIII transcription start and stop;
evidence for DROSHA processing?
GGGTCGAGCTGGACGGCGACGTAC
TTTTTCAGCTCGACCTGCCGCTGCATG
Where does DICER cut?
T
A
C
G
22 nts
Which strand goes into RISC?
(Strand that goes into RISC is more
stable/abundant)
TRC: Jen Grenier, Andrew Grimson, Ozan Alkan
Small RNA sequencing: all 26 shRNAs
Length#reads % shRNA % strand
GG
GG
GG
GG
23mer 5,217
22mer 32,279
21mer 8,029
20mer 1,029
G21merAntisenseStrandSTTTTT
(4)
(3)
A
(5)
T
11%
67%
17%
2%
C
G
GG21merSenseStrandSeqncC
1%
7%
2%
<1%
22mer
21mer
20mer
23mer
22mer
21mer
23mer
18,285
4%
39,095
9%
6,760
2%
45,610 10%
205,249 46%
40,444
9%
23,263
5%
5%
10% } 17%
2%
11%
51% } 72%
10%
6%
e
e
e
m
r
r
r
3Ts
4Ts
5Ts
3Ts
4Ts
5Ts
4Ts
Highly parallel identification of
essential genes in cancer cells
Biao Luo etc,
Proc Natl Acad Sci U S A, 2008, 105: 20380–20385.
Pooled RNAi screening (45K lentiviruses)
Pooled RNAi screening (45K lentiviruses)
Involed in
FAS induced
apoptosis
Screens for essential genes in 12 cancer cell lines
Commonly essential gene
NSCLC
glioblastoma
SCLC
leukemia
Cell lineage-specific essential gene
Cell line-specific essential gene
(Cancer specific gene dependency)
Time course analysis for the top
100 essential genes in K562 cells
Integration of functional and structural genomics
(Case in NSCLC)
Integration of functional and structural genomics
(Case in NSCLC)
Integration of functional and structural genomics
(Case in NSCLC)
References:
Review paper & original paper
1. Oncogene (2004) 23:8346-8352; 8376-8383; 8384-8391; 8392-8400; 8401-8409.
2. Moffat J & Sabatini DM 2006. Building mammalian signaling pathways with
RNAi screens. Nature Rev. 7:177-187.
3. Focus on RNA interference (a user guide). Nature Methods 2006 Sep 3(9):669-719.
4. Paddison PJ (2008) RNA interference in mammalian cell systems. Curr Top
Microbiol Immunol. 2008;320:1-19.
5. Recent reviews on RNAi. Curr Top Microbiol Immunol 2008, volume 320:1-201.
6. Luo Biao et al. 2008. Highly parallel identification of essential genes in cancer
cells. Proc Natl Acad Sci U S A. 105(51): 20380–20385.
National RNAi Core website
http://rnai.genmed.sinica.edu.tw/