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Silencing RNA techniques
- 1. Silencing RNA techniques (andCRISPR/Cas9 as well)… Kevin Petrie Wroclaw 16th September 2015
- 2. 2 Outline During RNAi Double-strandedRNAs 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
- 4. 4 RNAi: Silencing inCenorhabditis 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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- 7. 7 RNAi experiments Functional analysisby 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
- 8. 8 Strategy Optimized experiment: gene ofinterest knockdown Identify target gene of interest DsiRNA selection Cell line selection Optimize experimental conditions Controlled pilot experiment
- 9. 9 DsiRNA Processing Untreated controls siRNAtargeting 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 ofsplice 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
- 12. 12 If possible selectsequences 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 Designrules Design tools 13 Selecting an Effective siRNA
- 14. 14 Selecting an EffectivesiRNA DsiRNA selec on Design rules Design tools
- 15. 15 Experimental setup Additional parametersto 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
- 16. 16 Summary Uses: too manyto outline here! Include validation of on-target effects of drugs, control for ChIP, pathway analysis, functional screening etc
- 17. 17 shRNA Same principles applyas for siRNA Vector based: multiple options Transient, retroviral, lentiviral, inducible Beware so-called non-targeting controls – also perform empty vector and non-transfected controls
- 18. 18 CRISPR/CAS9: A bacterialimmune system We’ve learned how to highjack it!
- 19. 19 CRISPR/CAS9 S. Pyogenes cas9cuts 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
- 20. 20 CRISPR/CAS9 Using the cell’srepair 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
- 21. 21 CRISPR/CAS9 CRISPR is arapid and effective genome engineering method
- 22. 22 Generating CRISPR vectorsmade easy…
- 23. 23 Generating CRISPR vectorsmade easy…
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- 25. 25 Screening strategies shRNA: Mutiple options Highcost: eg Mission from Sigma Low cost: DECIPHER CRISPR
- 26. 26 Screening strategies Mohr etal, Nat Rev Mol Cell Biol, 2014
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Editor's Notes
- #5 RNAi can be induced in C. elegans in three simple ways: Injection of dsRNA into the worm gonads Soaking the worms in dsRNA solution Feeding the worms engineered bacteria producing dsRNA
- #7 Small interfering RNAs that have an integral role in the phenomenon of RNA interference (RNAi), a form of post-transcriptional gene silencing RNAi: 21-25 nt fragments, which bind to the complementary portion of the target mRNA and tag it for degradation A single base pair difference between the siRNA template and the target mRNA is enough to block the process. Each strand of siRNA has: a. 5’-phosphate termini b. 3’-hydroxyl termini c. 2/3-nucleotide 3’ overhangs
- #16 Hypoxanthine-guanine phosphoribosyltransferase
- #20 HNH – target strand