We compared the single-repressor CRISPRi (KRAB-dCas9) and dual-repressor CRISPRi (KRAB-dCas9-MeCP2) systems by targeting the transcription start sites and known enhancers of three genes (MRPS23, SLC25A37 and FSCN1) with two gRNAs per targeted region. We synthesized gRNAs as top and bottom strand oligos (IDT) and cloned them into BsmBI-digested lentiGuideFE-Puro. We transduced the cells in biological triplicate with gRNA lentiviruses at a low MOI and after 24 hours selected for cells with gRNAs using puromycin (1 μg/μL, Thermo Fisher A1113803). We harvested the cells 10 days after transduction and extracted RNA using TRIzol (ThermoFisher 15596026). We quantified RNA concentration by spectrophotometry (NanoDrop). To measure gene expression, we performed digital PCR (Formulatrix Consellation) with Cy5/Iowa Black RQ target gene probes (IDT), FAM/ZEN/Iowa Black FQ for the actin normalizer (IDT), and Luna Universal One-Step RT qPCR Master Mix kit (NEB E3005L) and Tween-20 (Sigma-Aldrich P1379). We first normalized the target gene expression by actin expression per sample and then normalized this ratio to the ratio from cells transduced with non-targeting control gRNAs.
Luna universal one step rt qpcr master mix kit
Manufactured by New England Biolabs
The Luna Universal One-Step RT qPCR Master Mix kit is a reagent designed for reverse transcription and quantitative real-time PCR (RT-qPCR) analysis. The kit contains all the necessary components for performing both reverse transcription and real-time PCR in a single reaction.
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2 protocols using luna universal one step rt qpcr master mix kit
1
Comparative CRISPRi Gene Repression Analysis
2
Comparative Analysis of CRISPRi Systems
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We compared the CRISPRi v1 and v2 systems by targeting the transcription start sites and known enhancers of three genes (MRPS23, SLC25A37 and FSCN1) with two gRNAs per targeted region. We synthesized gRNAs as top and bottom strand oligos (IDT) and cloned them into BsmBIdigested lentiGuideFE-Puro. We transduced the cells in biological triplicate with gRNA lentiviruses at a low MOI and after 24 hours selected for cells with gRNAs using puromycin (1 ug/uL, Thermo Fisher A1113803). We harvested the cells 10 days after transduction and extracted RNA using TRIzol (ThermoFisher 15596026). We quantified RNA concentration by spectrophotometry (NanoDrop). To measure gene expression, we performed digital PCR (Formulatrix Consellation) with Cy5/Iowa Black RQ target gene probes (IDT), FAM/ZEN/Iowa Black FQ for the actin normalizer (IDT), and Luna Universal One-Step RT qPCR Master Mix kit (NEB E3005L) and Tween-20 (Sigma-Aldrich P1379). We first normalized the target gene expression by actin expression per sample and then normalized this ratio to the ratio from cells transduced with non-targeting control gRNAs.
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