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Silencing RNA techniques(and CRISPR/Cas9 as well) Kevin PetrieWroclaw16th September 2015

2OutlineDuring 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 processVery potent activitySo far only been seen in eukaryotesEvidence 30% of genome is regulated by RNAi

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3OutlineRNA 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 MechanismDefense against Infection by viruses, etcAs a defense mechanism to protect against transposons and other insertional elements

Genome Wide RegulationRNAi plays a role in regulating development and genome maintenance. 30% of human genome regulated

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4RNAi: 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.

RNAi can be induced in C. elegans in three simple ways:Injection of dsRNA into the worm gonadsSoaking the worms in dsRNA solutionFeeding the worms engineered bacteria producing dsRNA

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5siRNA: Pathway

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6DsiRNA Processing

Small interfering RNAs that have an integral role in the phenomenon of RNA interference (RNAi), a form of post-transcriptional gene silencingRNAi: 21-25 nt fragments, which bind to the complementary portion of the target mRNA and tag it for degradationA 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 terminib. 3-hydroxyl terminic. 2/3-nucleotide 3 overhangs

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7RNAi experimentsFunctional 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 transcriptsiRNA selectionChoosing the cell type Validating the assayIncluding appropriate biological controls

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8Strategy

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9DsiRNA Processing

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10qPCR Assay Discordance: 2 Structure

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11Be careful of splice variants

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12If possible select sequences that work in human and mouse

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13Selecting an Effective siRNA

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14Selecting an Effective siRNA

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15Experimental setup

Additional parameters to optimize:Transfection reagentDose-response - reagentCell seeding densityDose-response DsiRNAForward/reverseTime courseReagent:DsiRNA ratioCell line validation:Literature searchExpression profileAssay validation:qPCR and Western

Hypoxanthine-guanine phosphoribosyltransferase 15

16Summary

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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17shRNASame principles apply as for siRNAVector based: multiple optionsTransient, retroviral, lentiviral, inducibleBeware so-called non-targeting controls also perform empty vector and non-transfected controls

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18CRISPR/CAS9: A bacterial immune systemWeve learned how to highjack it!

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19CRISPR/CAS9S. Pyogenes cas9 cuts genome upstream of NGG motif

Protospacer adjacent motif

HNH target strand19

20CRISPR/CAS9Using the cells repair pathways to engineer the genome

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21CRISPR/CAS9CRISPR is a rapid and effective genome engineering method

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22Generating CRISPR vectors made easy

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23Generating CRISPR vectors made easy

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24Screening strategies

shRNA orCRISPR

siRNA

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25Screening strategiesshRNA:Mutiple optionsHigh cost: eg Mission from Sigma

Low cost: DECIPHER

CRISPR

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26Screening strategies

Mohr et al, Nat Rev Mol Cell Biol, 2014

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27Screening strategiesOff-target effects: validate!

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