Gene editing workflow using the Strict-R™ Inducible Cas9 Lentiviral System

Gene knockout workflow using the Strict-R Inducible Cas9 System

Dharmacon™ Strict-R ™ Inducible Cas9 System demonstrates minimal leakiness across cell lines

Jurkat, U2OS, and A549 cells expressing Dharmacon™ Strict-R™ Inducible Hygromycin Cas9 Lentiviral system were transduced with Edit-R lentiviral sgRNA targeting CD71 (VSGH12605-257052386) or Edit-R lentiviral sgRNA non-targeting control #2 (VSGC12626) at MOI 0.3. After 24 hours, cells were selected with 2 μg/mL puromycin for 5 days. Jurkat cells were plated in 96-well plates at 50,000 cells/well, while U2OS and A549 were plated at 20,000 cells/well. All cells were stimulated with 500 ng/mL doxycycline and 500 nM Shield1, or left untreated (‘no induction’) for 6 days. On day 6, cells were collected and stained for receptor knockout analysis by flow cytometry. The lentiviral sgRNA transduced Jurkats, U2OS or A549 were stained with FITC anti-human CD71 (1:25). Non-targeting control transduced cells were used for positive control receptor stain and isotype controls to determine positive and negative receptor gates from the untreated media condition.

Dharmacon™ Strict-R™ Inducible Cas9 Lentiviral System performs as well or better than TRE3G-only regulated Edit-R Inducible Cas9

Jurkat cells expressing Edit-R™ Inducible Cas9 (hEF1a-Blast^R) Tet-only system or Dharmacon™ Strict-R™ Inducible Blasticidin- or Hygromycin-Cas9 Lentiviral System were transduced with Edit-R lentiviral sgRNA targeting CD28 (VSGH12605-256966516) or CD71 (VSGH12605-257052386) or Edit-R lentiviral sgRNA non-targeting control #2 (VSGC12626) at MOI 0.3. After 24 hours, cells were selected with 2 μg/mL puromycin for 5 days. Cells were plated in 96-well plates at 50,000 cells/well and stimulated with 500 ng/mL doxycycline and 500 nM Shield1, or left untreated (‘none’) for 6 days. On day 6, cells were collected and stained for receptor knockout analysis by flow cytometry. The lentiviral sgRNA transduced Jurkats were stained with PE anti-human CD28 antibody (1:100), or FITC anti-human CD71 (1:25). Non-targeting control Jurkat cells were used for positive control receptor stain and isotype controls to determine positive and negative receptor gates.

Gene editing activity of Strict-R Inducible Hygromycin-Cas9 Lentiviral System in A549 cells after induction with doxycycline + Shield1 for 72 hours

A549 were transduced with Dharmacon™ Strict-R™ Inducible Cas9 Lentiviral System with hygromycin resistance. After 14 days of hygromycin selection cells were transduced at an MOI of 0.3 with Edit-R pre-designed lentiviral sgRNA targeting PPIB positive control (VSGH12671), CD71/TFRC (VSGH12605-257052386) or CD58/LAF-3 (VSGH12605-256231081). Cells were selected for an additional 5 days with 2 μg/mL puromycin in TET-system approved A549 medium. For the gene editing experiment, double-selected A549 cell lines were plated in a 96-well plate at 20,000 cells/well in duplicate per induction condition: 500 ng/mL doxycycline + 500 nM Shield1, 500 ng/mL doxycycline alone, 500 nM Shield1 alone or no supplement to A549 medium (none). Cells were collected and lysed at 72 hours post induction to isolate genomic DNA for DNA mismatch detection assay with T7EI and Sanger sequencing with TIDE analysis. A. Gel electrophoresis (with FastRuler Low Range DNA Ladder) of T7EI endonuclease digested PCR products corresponding to gene target in A549. Below, in bar graph, is the quantification of T7EI gene editing by mismatch assay using the T7EI calculator. B. Quantification of gene editing by tracking of indels by decomposition (TIDE) analysis.

Robust gene editing and minimal leakiness with Dharmacon™ Strict-R™ Inducible Cas9 Lentiviral System in two cancer cell lines

A549 were transduced with Dharmacon™ Strict-R™ inducible Cas9 lentiviral system with hygromycin resistance. After 14 days of hygromycin selection cells were transduced at an MOI of 0.3 with Edit-R pre-designed lentiviral sgRNA targeting PPIB positive control (VSGH12671), CD71/TFRC (VSGH12605-257052386) or CD58/LAF-3 (VSGH12605-256231081). Cells were selected for an additional 5 days with 2 μg/mL puromycin in TET-system approved A549 medium. For the gene editing experiment, double-selected A549 cell lines were plated in a 96-well plate at 20,000 cells/well in duplicate per induction condition: 500 ng/mL doxycycline + 500 nM Shield1, 500 ng/mL doxycycline alone, 500 nM Shield1 alone or no supplement to A549 medium (none). Cells were collected and lysed at 72 hours post induction to isolate genomic DNA for DNA mismatch detection assay with T7EI and Sanger sequencing with TIDE analysis. A. Gel electrophoresis (with FastRuler Low Range DNA Ladder) of T7EI endonuclease digested PCR products corresponding to gene target in A549. Below, in bar graph, is the quantification of T7EI gene editing by mismatch assay using the T7EI calculator. B. Quantification of gene editing by tracking of indels by decomposition (TIDE) analysis

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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.