CRISPRi workflow using synthetic sgRNA and dCas9-SALL1-SDS3 lentiviral particles

Transduce cells with CRISPRi dCas9-SALL1-SDS3 lentiviral particles to generate CRISPRi-ready, stable dCas9-SALL1-SDS3 expressing cells. Then introduce CRISPRi synthetic sgRNA specific to your target gene(s). This system is ideal for arrayed screening in transfectable cell models.

CRISPRi workflow using lentiviral sgRNA and dCas9-SALL1-SDS3 lentiviral particles

Workflow using CRISPRi dCas9-SALL1-SDS3 lentiviral particles to establish a stable dCas9-SALL1-SDS3 expressing cell line, then delivering CRISPRi lentiviral sgRNA particles (left) or plasmid sgRNA (right). This system is ideal for working in difficult-to-transfect cell types.

CRISPRi with dCas9-SALL1-SDS3 results in more robust protein level repression than CRISPRi with dCas9-KRAB

Functional knockdown of TFRC was assessed in U2OS cells constitutively expressing either dCas9-KRAB or dCas9-SALL1-SDS3 under the hEF1α promoter. Cells were transfected with a 25 nM pool containing three CRISPRi synthetic sgRNAs targeting TFRC using DharmaFECT 4 Transfection Reagent. 72 hours post-transfection the cells were split. 96 hours post-transfection the cells were fixed and stained with a primary antibody targeting TFRC and an Alexa Fluor 488 conjugated fluorescent secondary antibody. Hoechst stain was used to identify nuclei.

Cells expressing dCas9-SALL1-SDS3 demonstrate greater and more consistent CRISPRi gene repression compared to dCas9-KRAB

CRISPRi synthetic sgRNAs achieve maximal repression at 48-72 hours post-transfection in both dCas9-KRAB and dCas9-SALL1-SDS3-expressing cells. U2OS cells stably expressing integrated dCas9-KRAB or dCas9-SALL1-SDS3 were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with pools of CRISPRi synthetic sgRNA (25nM) targeting CBX1, HBP1, or SEL1L. Cells were harvested at 24, 48, 72, 96, 120, and 144 hours post-transfection, total RNA was isolated, and relative gene expression was measured using RT-qPCR. The relative expression of each target gene was calculated with the Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control (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.