Gene repression workflow using the Dharmacon™ Strict-R™ Inducible CRISPRi Lentiviral System

Gene repression workflow using the Strict-R Inducible CRISPRi Lentiviral System

Robust transcriptional repression and minimal leakiness with the Dharmacon™ Strict-R™ Inducible CRISPRi Lentiviral System across cell lines

U2OS, K562, and Jurkat cells expressing either the Dharmacon™ Strict-R™ Inducible CRISPRi Lentiviral System (green) or dCas9-SALL1-SDS3 under control of the listed constitutive promoter (purple; hEF1α, mCMV, or hCMV, selected for optimal activity in the respective cell line) were transduced with CRISPRmod CRISPRi lentiviral sgRNA targeting PPIB or SEL1L (labeled as targeting sgRNA +) or a CRISPRmod CRISPRi lentiviral non-targeting control (labeled as NTC +) at MOIs of 0.3. After 48 hours, cells were selected with 2.5 µg/µL puromycin for 5 to 7 days. Cells were plated in 96-well plates at 20,000 cells/well and stimulated with 0.5 µg/ mL doxycycline and 500 nM Shield1 for 48 hours prior to harvest. Total RNA was isolated and relative gene expression was measured using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to an NTC.

Potent, reversible gene repression with the Dharmacon™ Strict-R™ Inducible CRISPRi Lentiviral System in K562 cells

K562 cells expressing the Dharmacon™ Strict-R™ Inducible CRISPRi Lentiviral System were transduced with CRISPRi lentiviral sgRNA targeting PPIB or a CRISPRi lentiviral non-targeting control (NTC) at MOIs of 0.3. After 48 hours, cells were selected with 2.5 µg/µL puromycin for 5 days. Cells were plated in 6-well plates at 200,000 cells/well and stimulated with 0.5 µg/ mL doxycycline and 500 nM Shield1 with small molecule-containing media replenished every 48 hours. Populations only receiving an initial pulse were washed with PBS after 48 hours and subsequently cultured in media without the listed small molecules for the remainder of the experiment. At each time point, aliquots of cells were harvested, total RNA was isolated, and relative gene expression was measured using RT-qPCR. The relative expression of PPIB was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to an NTC.

Raising Shield1 concentrations up to 500 nM result in more robust gene repression with the Dharmacon™ Strict-R™ Inducible CRISPRi Lentiviral System

U2OS cells expressing the Dharmacon™ Strict-R™ Inducible CRISPRi Lentiviral System were transduced with CRISPRi lentiviral sgRNA targeting PPIB or SEL1L, or a CRISPRi lentiviral non-targeting control (NTC) at MOIs of 0.3. After 48 hours, cells were selected with 2.5 µg/µL puromycin for 5 days. Cells were plated in 96-well plates at 10,000 cells/well and stimulated with media containing 0.5 µg/ mL doxycycline and the listed concentrations of Shield1. After 48 hours, cells were harvested, total RNA was isolated, and relative gene expression was measured using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to an NTC.

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Supporting Data

Improved CD8+ T-cell SMARTvector™ shRNA lentiviral system transduction using LentiBOOST™ Lentivirus Transduction Enhancer

100,000 primary human CD8+ T cells were transduced with either 30,000 (MOI 3, green) or 70,000 (MOI 7, purple) TUs of SMARTVector™ mCMV tGFP Lentiviral Control Particles targeting either NTC or PPIB along with 1:100 LentiBOOST transduction enhancer. Cells were centrifuged at 800 x g for one hour at 32 °C followed by a four hour incubation prior to removal of lentiviral particles and transduction enhancer. Transduction efficiency (%GFP+ out of live cells) and viability were determined 5 days post-transduction by flow cytometry. The addition of LentiBOOST technology markedly improved transduction efficiencies without significantly impacting cell viability.

Improved CD4+ and CD8+ T-cell Edit-R™ All-in-one sgRNA/Cas9 lentiviral system transduction using LentiBOOST™ Lentivirus Transduction Enhancer

100,000 primary human CD4+ and CD8+ T cells from two donors were transduced with 250,000 TUs of Edit-R GFP Delivery controls mCMV along with 1:100 LentiBOOST transduction enhancer. Cells were centrifuged at 800 x g for one hour at 32 °C followed by an overnight incubation prior to removal of lentiviral particles and transduction enhancer. Transduction efficiency and viability were determined 72 hours post-transduction by flow cytometry. The addition of LentiBOOST technology markedly improved transduction efficiencies without significantly impacting cell viability.

Improved transduction of human induced pluripotent stem cells (hiPSCs) with the Strict-R™ Inducible CRISPRa lentiviral system transduction using LentiBOOST™ Lentivirus Transduction Enhancer

10,000 WTC hiPS cells were transduced with either 20,000 (MOI 2, green) or 40,000 (MOI 4, purple) TUs of Strict-R™ Inducible EGFP dCas9-VPR Lentiviral Particles along with 1:100 LentiBOOST transduction enhancer. Cells were centrifuged at 800 x g for one hour at 32 °C followed by an overnight incubation prior to removal of lentiviral particles and transduction enhancer. Transduction efficiency and viability were determined 72 hours post-transduction by flow cytometry. The addition of LentiBOOST markedly improved transduction efficiencies without significantly impacting cell viability.