Gene editing workflows for lentiviral Cas9 nuclease reagent formats

Gene knockout workflow using the lentiviral Cas9 nuclease with Edit-R lentiviral sgRNA

Gene knockout workflow using the lentiviral Cas9 nuclease with sgRNA system. Gene editing can be performed using a mixed cell population approach (left side) typically for gene knockout screening or an isolated clonal cell line approach (right side) when a more defined cell type is required for phenotypic analysis.

Gene knockout workflow using the inducible lentiviral Cas9 nuclease with Edit-R lentiviral sgRNA

Gene knockout workflow using the lentiviral Cas9 nuclease with Edit-R synthetic guide RNA

Gene knockout workflow using the inducible lentiviral Cas9 nuclease in a pooled lentiviral sgRNA screen

Lentiviral fluorescent Cas9 nuclease data

Fluorescent lentiviral Cas9 nuclease enables enrichment for high expression and improved CRISPR-Cas9 gene editing efficiency

U2OS cells were transduced at low multiplicity of infection (MOI 0.3) with Edit-R Lentiviral hCMV mKate2-Cas9 Nuclease particles (Cat #VCAS11869) so that transduced cells would have only one integration of Cas9. Cells were expanded for fluorescence activated cell sorting (FACS) where populations were sorted into negative, low, medium and high mKate2 fluorescence. These subpopulations were expanded and then plated at 10,000 cells/well in a 96-well plate. One day later, cells were transfected with tracrRNA (25 nM, Cat #U-002005-xx) and Edit-R PPIB Synthetic crRNA Control (Cat #U-007501-xx) or Edit-R MYC Predesign crRNA (Cat #CM-003282-01) using DharmaFECT 1 transfection reagent (0.3 µL/well, Cat #T-2001-01). After 72 hours, cells were imaged for mKate2 fluorescence using the In Cell Analyzer 2200 (GE Healthcare; A) and then harvested for DNA mismatch detection assay to estimate gene editing (B). High mKate2 expression can be associated with the highest levels of gene editing for both PPIB- and MYC-targeting crRNAs.

Lentiviral inducible Cas9 nuclease data

Gene editing activity of inducible Cas9 vectors after induction with Doxycycline for 7 days

Cells were transduced with a constitutive (CAG-Cas9) or an inducible (TRE3G-Cas9) Cas9 expression lentiviral particles at an MOI of 0.3, and selected with 10 µg/mL blasticidin in tetracycline-free medium for 10 days. Cas9-stable cells were then transduced with DNMT3B- or PPIB-sgRNA lentiviral particles at an MOI of 0.3. Cells were selected with 2 µg/mL puromycin for 2 days in tetracycline-free medium and split in two populations: uninduced (DOX-) and induced (DOX+) with 500 ng/mL doxycycline for 7 days. The cells were then lysed and analyzed for indels using a DNA mismatch detection assay with T7EI.

Inducible Cas9 nuclease vector displays minimal leak after 21 days without doxycycline induction

Cells were transduced with a constitutive (CAG-Cas9) or an inducible (TRE3G-Cas9) Cas9 expression lentiviral particles at an MOI of 0.3, and selected with 10 µg/mL blasticidin in tetracycline-free medium for 10 days. Cas9-stable cells were then transduced with DNMT3B- or PPIB-sgRNA lentiviral particles at an MOI of 0.3. Cells were selected with 2 µg/mL puromycin and maintained in tetracycline-free medium for 21 days. The cells were then lysed and analyzed for indels using a DNA mismatch detection assay with T7EI.

Dose response for doxycycline in inducible U2OS-Cas9 cells

Cells were transduced with the inducible (TRE3G-Cas9) Cas9 expression lentiviral particles at an MOI of 0.3, and selected with 10 µg/mL blasticidin in tetracycline-free medium for 10 days. Cas9-stable cells were then transduced with PPIB-sgRNA lentiviral particles at an MOI of 0.3. Cells were selected with 2 µg/mL puromycin tetracycline-free medium for 4 days, suspended with trypsin and seeded in a 96-well plate in medium containing increasing concentrations of doxycycline (0 to 1000 ng/mL). The cells were incubated for 72 hours, lysed and analyzed for indel formation using a DNA mismatch detection assay with T7EI. Upper panel, representative gel image of the DNA mismatch detection assay with T7EI for PPIB targeted amplicon; lower panel, mean ± standard deviation of the estimated percentage of gene editing from three independently treated wells.

Lentiviral Cas9 nuclease data

Differential expression of Cas9 by different promoters induce varying levels of gene editing

A human recombinant U2OS ubiquitin-EGFP proteasome cell line (Ubi[G76V]-EGFP) (A) and a mouse fibroblast (NIH/3T3) (B), were stably transduced with lentiviral particles containing Cas9 and a blasticidin resistance gene driven by the indicated promoters.. A population of cells with stably integrated Cas9-blastR was selected with blasticidin for a minimum of 10 days before transfections. Cells were transfected with 50 nM synthetic crRNA:tracrRNA targeting Human PPIB / mouse Ppib using DharmaFECT 1 and DharmaFECT 3 Transfection reagent, respectively. After 72 hours, the relative frequency of gene editing was calculated based on a DNA mismatch detection assay using T7EI on genomic DNA extracted from the transfected cells.

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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.