The Edit-R guide RNA guarantee

We guarantee that EVERY predesigned Edit-R 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.

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 Cas9 nuclease mRNA and synthetic sgRNA or crRNA:tracrRNA

 

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 Gene knockout workflow using Cas9 nuclease protein and synthetic sgRNA or crRNA:tracrRNA

 

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Gene knockout workflow using lentiviral Cas9 expression particles and synthetic sgRNA or crRNA:tracrRNA

Complete your knockout experiment by adding these supporting reagents to your order

Cas9 nuclease options

• Cas9 mRNA or Cas9 protein NLS can be co-transfected with Edit-R synthetic sgRNA for a fully DNA-free workflow.
• Stable Cas9 expressing cell lines are ready to be transfected with Edit-R synthetic sgRNA for rapid gene knockout.

Synthetic guide RNA controls

• Non-targeting controls are used to evaluate cellular responses to CRISPR-Cas9 components in the absence of gene target-specific sgRNA. Choose from 5 scrambled sequences guaranteed not to target any gene in the human or mouse genomes.

• Cutting controls

  Cutting control (safe harbor) synthetic sgRNAs recommended for determination of baseline cellular responses in CRISPR-Cas9 experiments.

• Positive controls targeting well-characterized genes are used to determine the effectiveness of your gene editing conditions for maximal efficiency. Choose from PPIB or DNMT3B.

Transfection reagents

• DharmaFECT transfection reagents are optimized for improved delivery and reduced toxicity. The optimal DharmaFECT reagent for your experiment will depend on your Cas9 nuclease source and your cell type.

Cas9 nuclease source	Recommended transfection reagent
Stable Cas9 expressing cell line or Cas9 nuclease lentiviral particles	DharmaFECT 1,2,3, or 4 for transfection of synthetic guide RNA. Use this guide to find the recommended DharmaFECT reagent for your cell type.
Cas9 mRNA or protein NLS	DharmaFECT Duo for co-transfection of mRNA or protein and synthetic guide RNA

Flow cytometry analysis of protein knockout in CD4⁺ T cells using a single predesigned synthetic sgRNA per gene target

Primary human CD4⁺ T cells were nucleofected with Cas9 RNP using an individual predesigned synthetic sgRNA per gene target or a non-targeting control (NTC), via a Lonza 96-well Shuttle system. After 72 hours, functional knockout of CD28, CD3E, and CXCR3 were assessed as a percent of cells not expressing the target gene by FACS analysis. Cells were stained and normalized for CD4 expression using an Alexa Fluor 488 conjugated antibody and compared to each target CD28, CD3E, and CXCR3 using APC conjugated primary antibodies.

 

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Functional knockout of FUS gene with a pool of synthetic sgRNA

Functional knockout of FUS was assessed in U2OS cells constitutively expressing Cas9 under the CAG promoter. Cells were transfected with a 25 nM sgRNA pool containing three different Edit-R predesigned sgRNAs targeting FUS using 0.04 µL DharmaFECT 4. 72 hours post transfection the cells were split, and at 96 hours post transfection, cells were fixed and stained with a primary antibody targeting FUS, and an Alexa Fluor 488 conjugated fluorescent secondary antibody. Hoechst stain was used to identify nuclei.

 

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Nuclease stabilization modifications improve gene editing efficiency

HEK293T and U2OS integrated Cas9 (under the CAG promoter) cell lines were transfected with unmodified and 5’ and 3’ 2xMS modified synthetic sgRNA targeting PPIB and DNMT3B genes. At 72 hours editing efficiency was assessed with a mismatch detection assay, T7 Endonuclease I (T7EI). In all cases the stabilizing modifications improve gene editing efficiency. MS = 2’-O-methyl nucleotides and phosphorothioate backbone linkage.

 

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Edit-R synthetic guide RNAs cause virtually no innate immune response or toxicity compared to in vitro transcribed guide RNA

A HEK293T Cas9 nuclease expressing cell line was transfected with different synthetic guide RNA formats including unmodified crRNA:tracrRNA, crRNA:tracrRNA modified with 2xMS on 5’ crRNA and 3’ tracrRNA, crRNA:tracrRNA modified with 3xMS on both 5’ and 3’ crRNA and tracrRNA, unmodified synthetic sgRNA, modified synthetic sgRNA with 5’ and 3’ 2xMS or 3xMS, and in vitro transcribed (IVT) sgRNA targeting PPIB and DNMT3B genes. At 72 hours viability was assessed with the Resazurin reduction assay (red dots) and the levels of five immune response genes were quantified by RT-qPCR. MS = 2’-O-methyl nucleotides and phosphorothioate backbone linkages.

 

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Structure of modifications for improved nuclease resistance on Edit-R synthetic sgRNA

Modifications for improved nuclease resistance: 2’-O-methyl modified nucleotides and phosphorothioate backbone linkages.

 

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Sequence structure of Edit-R synthetic single guide RNA

The structure of a Edit-R synthetic single guide RNA showing the 2xMS nuclease stability modifications on both 5’ and 3’ ends of the molecule. The structure contains a 20 nt targeting sequence (shown in green as poly Ns), 12 nt crRNA repeat sequence (shown in light gray), 4 nt tetraloop sequence (shown in bold black), and a 64 nt tracrRNA sequence (shown in blue). MS = 2’O-methyl nucleotides and phosphorothioate linkages (MS).

How much sgRNA do I need?

This table provides the approximate number of experiments that can be carried out for lipid transfection methods at the recommended sgRNA working concentration (25nM) in various plate/well formats. Calculations do not account for pipetting errors.

sgRNA nmol	96-well plate 100 µL reaction volume	24-well plate 500 µL reaction volume	12-well plate 1000 µL reaction volume	6-well plate 2500 µL reaction volume
2	800	160	80	32
5	2000	400	200	80
10	4000	800	400	160

1. R. Barrangou, A. Birmingham, et. al. Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference. Nucleic Acids Research, 43(7) 3407-3419 (2015)
2. M.L. Kelley, Ž. Strezoska, et al. Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing. J. Biotechnol. 233, 74–83 (2016). doi:10.1016/j.jbiotec.2016.06.011
3. Basila, M., M. L. Kelley, et al.. Minimal 2’-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity. PLoS One. 12, e0188593 (2017). doi: 10.1371/journal.pone.0188593
4. Anderson, E.M., A. Haupt, et al. Systematic analysis of CRISPR-Cas9 mismatch tolerance reveals low levels of off-target activity. J. Biotechnol. 211, 56-65 (2015)
5. He, K., E. Chou, et al. Conjugation and evaluation of triazole-linked single guide RNA for CRISPR-Cas9 gene editing. ChemBioChem. DOI: 10.1002/cbic.201600320 (2016)