The Edit-R predesigned guide RNA guarantee

We guarantee that EVERY predesigned guide RNA will provide successful editing at the target site when delivered as described in the Edit-R Technical Manuals.

The Edit-R guide RNA guarantee is valid when used with any wild type S. pyogenes Cas9 nuclease, including mRNA, expression plasmid, protein, or stable Cas9 expression, and Edit-R crRNAs must be used with Edit-R tracrRNA for the guarantee to apply.

Analysis of editing of the treated cell population must be shown using a T7EI or Surveyor mismatch detection assay. If successful editing is not observed for a predesigned Edit-R guide RNA while an appropriate side-by-side Edit-R positive control is successful, a one-time replacement of a different predesigned Edit-R guide RNA of the same format and quantity will be provided at no cost.

A replacement will only be approved upon discussion with our Scientific Support team.

Successful editing at the DNA level does not always lead to functional gene knockout; it is recommended to test multiple guide RNAs to determine the most effective guide RNA for knockout of your target gene.

This guarantee does not extend to any accompanying experimental costs, does not apply to guide RNAs ordered via the CRISPR Design Tool, and will not be extended to the replacement guide RNA.

Gene knockout workflow using the Edit-R All-in-one lentiviral sgRNA system

Gene knockout workflow with the All-in-one lentiviral sgRNA using a mixed population (left side) or clonal cell line (right side) experimental approach. If a homogeneous gene reduction across the entire population is required, then the clonal isolation workflow is recommended.  Additionally of note, if knocking out the gene-of-interest is lethal or inhibits cell growth, the post-selection cells likely will retain wild-type or have an in-frame mutation that doesn't affect downstream protein expression. 

Edit-R all-in-one lentiviral sgRNA controls

• Positive controls and detection primers

  Control all-in-one lentiviral sgRNAs to verify DNA double-strand breaks and gene editing efficiencies.

• Non-targeting controls

  All-in-one lentiviral sgRNA constructs bioinformatically designed and validated to not target any gene in human, mouse or rat genomes.

• Cutting controls

  Lentiviral sgRNA constructs targeting inactive “safe harbor” genomic regions in the human and mouse genomes.

Edit-R All-in-one lentiviral system percent editing (TIDE)

Jurkat or K562 cells were transduced at a MOI of 0.3 using Edit-R All-in-one EGFP Lentiviral sgRNA vectors targeting either the CD3 or CD28 genes. All-in-one lentiviral particles express Cas9 nuclease constitutively under the hEF1α or mCMV promoters. Lentiviral particles expressing Non-targeting control (NTC) guides were used as negative controls. After lentiviral transduction and expansion, cells were analyzed as unsorted population (pool) or were subjected to cell sorting to enrich for the brightest (top) or dimmest (dim) EGFP expressing cell populations. The pool, top, and dim populations of cells were then subjected to TIDE analysis to assess gene editing frequency.

Comparison of Edit-R two-vector and All-in-one systems

For the two-vector system, cells were previously transduced with a constitutive hEF1α- or mCMV-Cas9 expression lentiviral particles at an MOI of 0.3, and selected with 10 µg/mL blasticidin for 10 days, followed by transduction with non-targeting control (NTC), DNMT3B- or PPIB-sgRNA lentiviral particles at an MOI of 0.3. For the All-in-one system, wild-type cells were transduced with the NTC, DNMT3B or PPIB All-in-one lentiviral particles that also express a constitutive hEF1α- or mCMV-Cas9 nuclease, and the Puro^R gene at an MOI of 0.3. All transduced cells were selected with 2 µg/mL puromycin for 3 days. The cells were then lysed and analyzed for indels using a DNA mismatch detection assay with T7EI.

Schematic diagram of the Edit-R All-in-one lentiviral sgRNA vector

In the Edit-R All-in-one lentiviral sgRNA vector backbone, the gene-specific guide RNA is expressed under the control of a human U6 promoter, while expression of the Cas9 and puromycin resistance marker (Puro^R) or EGFP marker is driven from either the human EF1α or the mouse CMV promoter. The plasmid contains the Amp^R resistance marker for growth and selection in E. coli.

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.