This document discusses RNA interference (RNAi) techniques such as silencing RNA and CRISPR/Cas9. It explains that double-stranded RNA is cut by the enzyme Dicer into short interfering RNAs (siRNAs) that can degrade mRNA strands in a highly specific process. RNAi is involved in regulating 30% of the human genome and acts as a defense mechanism against viruses and transposons. The document also discusses selecting effective siRNAs, considerations for species variation and secondary RNA structures, and strategies for gene knockdown screening using shRNA and CRISPR/Cas9.

1. Silencing RNA techniques
(and CRISPR/Cas9 as well)…
Kevin Petrie
Wroclaw
16th September 2015

2. 2
Outline
During RNAi Double-stranded RNAs cut into short double-stranded RNAs,
s(small) i(interfering) RNA's, by an enzyme called Dicer. These then base pair to
an mRNA through a dsRNA-enzyme complex. This will either lead to degradation
of the mRNA strand.
• Highly specific process
• Very potent activity
• So far only been seen in eukaryotes
• Evidence 30% of genome is regulated by RNAi

3. 3
Outline
RNA interference (RNAi) is a mechanism that inhibits gene expression at the
stage of translation or by hindering the transcription of specific genes.
RNAi targets include RNA from viruses and transposons.
Defense Mechanism
Defense against Infection by viruses, etc
As a defense mechanism to protect against transposons and other insertional
elements
Genome Wide Regulation
RNAi plays a role in regulating development and genome maintenance.
30% of human genome regulated

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RNAi: Silencing in Cenorhabditis elegans
• dsRNA administrated to worms can permeate and affect the entire body
causing a systemic RNA-interference
• RNAi studies represents a means of identifying partial or complete loss-of-
function phenotypes, possibly leading to the identification of gene function.

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siRNA: Pathway

6. 6
DsiRNA Processing

7. 7
RNAi experiments
Functional analysis by mRNA knockdown using siRNAs or shRNAs is now
routine in many molecular biology labs. However, many RNAi-related
experiments fail due to diversion from simple, good practices.
Steps leading to successful siRNA experiments include:
• Understanding the target transcript
• siRNA selection
• Choosing the cell type
• Validating the assay
• Including appropriate biological controls

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Strategy
Optimized experiment:
gene of interest
knockdown
Identify target gene of interest
DsiRNA selection
Cell line selection
Optimize experimental conditions
Controlled pilot experiment

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DsiRNA Processing
Untreated controls
siRNA targeting gene of
interest
Cycle
∆Rn
∆ Cq > 3.3, 90% knockdown
Amplification Plot
qPCR – Gene of Interest (GOI) Expression in HeLa Cells

10. 10
qPCR Assay Discordance: 2º Structure
Identify target
gene of interest
2° Structure
Transcript
variants
Species
variation
Assay discordance appears at the
3’-end of the transcript.
Measured mRNA levels show
significant divergence
Retained mRNA fragments
“Geographically” spaced qPCR
assays
0
10
20
30
40
50
60
70
80
90
100
110
120
0 500 1000 1500
mRNARemaining(%)
GOI Knockdown in HeLa Cells at 1 nM
Normalized to Hs HPRT and SFRS9 vs NC1, NC5, and NC7
Assay 1 Assay 2 5' UTR CDS 3' UTR

11. 11
Be careful of splice variants…
qPCR Assay Loc DsiRNALoc
Identify target
gene of interest
2° Structure
Transcript
variants
Species
variation
Transcript variants (and relative abundance) can affect results in a qPCR assay and DsiRNA location-
dependent fashion.
1
2
3
4
5

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If possible select sequences that work in human and mouse
Identify target
gene of interest
2° Structure
Transcript
variants
Species
variation
Hs GOI
Mm GOI
Region of Mm/Hs sequence homology
Interspecies alignment of mRNA sequence can affect future experimental directions.

13. DsiRNA selec on
Design rules
Design tools
13
Selecting an Effective siRNA

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Selecting an Effective siRNA
DsiRNA selec on
Design rules
Design tools

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Experimental setup
Additional parameters to optimize:
Transfection reagent
Dose-response - reagent
Cell seeding density
Dose-response – DsiRNA
Forward/reverse
Time course
Reagent:DsiRNA ratio
Cell line validation:
Literature search
Expression profile
Assay validation:
qPCR and Western

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Summary
Uses: too many to outline here!
Include validation of on-target effects of drugs, control for ChIP,
pathway analysis, functional screening etc

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shRNA
Same principles apply as for siRNA
Vector based: multiple options
Transient, retroviral, lentiviral, inducible
Beware so-called non-targeting controls
– also perform empty vector and non-transfected controls

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CRISPR/CAS9: A bacterial immune system
We’ve learned how to highjack it!

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CRISPR/CAS9
S. Pyogenes cas9 cuts genome upstream of “NGG” motif
September 15 9
Two cu ng domains:
• HNH
• RuVC
Constant scaffold for
Cas9 binding
Jinek et al.,Science 2012
Hsu et al.,Nature 2013
Protospacer adjacent motif

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CRISPR/CAS9
Using the cell’s repair pathways to engineer the genome
HI GH FREQUENCY
ERROR-PRONE
LOW FREQUENCY
HI GH FI DELI TY
Non Homologous
End Joining (NHEJ)
Homology Directed
Repair (HDR)
Double Stranded Break

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CRISPR/CAS9
CRISPR is a rapid and effective genome engineering method

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Generating CRISPR vectors made easy…

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Generating CRISPR vectors made easy…

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Screening strategies
shRNA or
CRISPR
siRNA

25. 25
Screening strategies
shRNA:
Mutiple options
High cost: eg Mission from Sigma
Low cost: DECIPHER
CRISPR

26. 26
Screening strategies
Mohr et al, Nat Rev Mol Cell Biol, 2014

27. 27
Screening strategies
Off-target effects: validate!