CRISPRi lentiviral sgRNA workflow

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).

CRISPRi lentiviral sgRNA controls

CRISPRi lentiviral sgRNA positive controls

• CRISPRi sgRNAs targeting well-characterized genes to determine the effectiveness of your experimental conditions for maximum repression.

CRISPRi lentiviral sgRNA non-targeting controls

• CRISPRi non-targeting controls to evaluate baseline cellular responses to CRISPRi components in the absence of target-specific sgRNA.

Related products

CRISPRi dCas9-SALL1-SDS3

• Lentiviral particles or mRNA that express nuclease-deactivated Cas9 fused to our proprietary transcriptional repressors for CRISPRi experiments.

CRISPRi lentiviral sgRNA is well suited for long term repression

U2OS and A549 cells stably expressing integrated dCas9-KRAB or dCas9-SALL1-SDS3 were plated at 50,000 cells/well in 24 well plates and transduced with CRISPRi sgRNA expressing lentiviral particles targeting PPIB or SEL1L at a MOI of 0.3 to obtain cells with a single integrant. Cells were selected with 2 µg/mL puromycin for 5 days, split into two populations, and allowed to recover for an additional day. At 7 days post-transduction one population was harvested for RT-qPCR analysis. The replicate populations of cells were cultured for 7 additional days before harvest. Total RNA was isolated from harvested plates and relative gene expression was calculated with the Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.

1. M. A. Horlbeck et al., Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife. 5, e19760 (2016).

LentiBOOST Lentivirus Transduction Enhancer is a uniquely formulated transduction reagent that can be used with or without lentivirus spinfection in order to increase successful viral transduction events while preserving cell viability. Especially critical for preserving precious primary cells from patient cohorts, or, for engineering complex animal models; improving transduction efficiency can save time and costs by increasing the success of each editing/transduction step, or, even avoid the loss of irreplaceable samples. Additionally, LentiBOOST technology is already used in the manufacturing of a number of clinical stage therapies providing the opportunity to demonstrate improved workflow applicability to the clinic.

LentiBOOST can be purchased through the Dharmacon Reagents catalog.

To learn more about LentiBOOST technology visit the Revvity LentiBOOST webpage.

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.