Functional and specific targeting for high-confidence gene knockout results
CRISPR-Cas9 is a highly effective tool for interrogating gene function, yet not all guide RNAs (gRNA) are effective in attaining functional protein knockout. To address this problem, Dharmacon developed an algorithm that is trained and validated for functional knockout, not just the ability to create double-strand breaks (DSB). With pre-designed CRISPR RNA (crRNA) you can quickly and easily assess multiple crRNA per gene, for many different genes.
Edit-R crRNA is a chemically synthesized RNA, comprised of 20 nucleotides identical to the genomic DNA target site, or protospacer, followed by the required S. pyogenes repeat sequence that interacts with the tracrRNA, which is required for use with synthetic crRNA. The chosen 20-base target sequence in the gene is immediately upstream of a protospacer-adjacent motif (PAM) in the genomic DNA.
Chemical modifications are applied to all predesigned crRNAs to resist degradation by nucleases to improve performance in applications using co-delivery with DNA-free Cas9. Learn more in this featured article.
100% of Edit-R predesigned guide RNA are guaranteed to edit!
Edit-R predesigned crRNA and lentiviral sgRNAs are guaranteed to edit your target, or we will replace it! No restrictions on Cas9 nuclease formats – if your Edit-R positive control works, so will your gene-specific guide RNA. Click on the Specifications tab to learn more about our guarantee.
Required componentsfor an Edit-R CRISPR-Cas9 gene engineering experiment using synthetic crRNA:
How much crRNA & tracrRNA do I need?
This table provides the approximate number of experiments that can be carried out for lipid transfection methods at the recommended crRNA:tracrRNA working concentration (25 nM:25nM) in various plate/well formats. Calculations do not account for pipetting errors.
100 µL reaction volume
500 µL reaction volume
1000 µL reaction volume
2500 µL reaction volume
The Edit-R Predesigned Guide RNA Guarantee
We guarantee that EVERY predesigned Edit-R CRISPR-Cas9 crRNA and Edit-R lentiviral sgRNA (guide RNAs) 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 T7E1 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 Technical Support team (firstname.lastname@example.org).
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.
crRNA with high scores from the Edit-R algorithm have higher cleavage efficiency than low-scoring designs
10 crRNAs with high functional scores for 10 genes (blue bars) and 10 crRNAs with low functional scores for the same genes (yellow bars) were tested for editing by Next Generation Sequencing. 93% of the high-scoring crRNAs and 32 % of the low scoring crRNAs showed > 40% of editing (indel formation). The Cas9-HEK293T cell line was transfected with 50 nM crRNA:tracrRNA, using 0.25 µL/well of DharmaFECT 1. Seventy-two hours post-transfection, cells were lysed and Nextera transposon-adapted amplicons spanning each crRNA site were generated for every treated sample as well as for a matched control amplicon from untransfected samples. Samples were indexed using the Nextera 96-well index kit and pooled for sequencing on a MiSeq instrument (paired end reads, 2 x 300 length). Reads that passed NGS quality filtering criteria were aligned to the reference file (Bowtie2 v2.1.0). Percent perfect reads were calculated and normalized to the control untransfected samples (Samtools v0.1.12a); the data is presented as normalized percent edited.
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.
Position and structure of modifications for improved nuclease resistance on Edit-R synthetic crRNA and tracrRNA
A. Position of modifications for nuclease resistance on Edit-R synthetic crRNA and tracrRNA. Edit-R synthetic guide RNAs contain 2ʹ-O-methyl (2ʹ-OMe) and backbone phosphorothioate linkages (PS) on the two nucleotides at the 5ʹ end of the crRNA (green strand) and on the two nucleotides at the 3’ end of the tracrRNA (blue strand). B. Structure of the 2ʹ-OMe and PS backbone phosphorothioate linkages.
Synthetic crRNA:tracrRNA is compatible with all Cas9 Nuclease formats
U2OS cells were plated at 10,000 cells/well one day prior to transfection. Cells were transfected with either Edit-R Cas9 Nuclease plasmid (200 ng), Edit-R Cas9 Nuclease mRNA(200 ng) or Cas9 nuclease protein (25 nM) and crRNA:tracrRNA (25 nM) targeting PPIB using DharmaFECT Duo transfection reagent (0.4 uL/well) in biological triplicates.
Efficiency of gene editing with 2x MS modified or unmodified crRNA:tracrRNA
A. Modifications to block degradation by nucleases are required for successful co-electroporation of Cas9 mRNA and crRNA:tracrRNA. B. Co-electroporation of Cas9 protein and crRNA:tracrRNA does not require stabilizing modifications, but results may be improved with their addition.
Efficient gene editing in Cas9-expressing NIH/3T3 cell lines by transfection with Edit-R crRNA:tracrRNA
NIH/3T3 cells were stably transduced with lentiviral particles containing Cas9 and a blasticidin resistance gene driven by the indicated promoters. A population of stably integrated cells were selected with blasticidin for a minimum of 10 days before transfections. Cells were transfected with 50 nM Edit-R synthetic crRNA:tracrRNA targeting PPIB using DharmaFECT 1 transfection reagent. After 72 hours, the relative frequency of gene editing was calculated based on a DNA mismatch detection assay with T7 Endonuclease I.
crRNAs with high functional scores from the Edit-R algorithm show stronger phenotypes in ApoONE assay than low-scoring designs
U2OS-Proteasome cells with integrated Cas9 (under CAG promoter) were plated in 96-well plates at 10,000 cells per well. 24 h after plating, cells were transfected with 25 nM crRNA:tracrRNA using 0.2 µg/well of DF4. Cell were analyzed for apoptosis 48 h after transfection using the ApoONE homogeneous assay (Promega). For the box plot, the crRNAs were divided into bottom half (H1) and top half (H2) based on their Edit-R algorithm functional score (110 total data points represented). The medians, distribution of data between the lower and upper halves and the minimum and maximum values demonstrate that high-scoring crRNAs have increased functionality.
Designs with gaps & mismatches can cause off-target cleavage
T7EI mismatch analysis for a crRNA showing the intended target site (GGTCATCTGGGAGAAAAGCG) and a predicted off-target site that was identified by the Edit-R alignment tool but no other online tool, containing one gap and one mismatch (GGT-ATCTGGGAGAAAAGCa) Many commonly used web-based crRNA specificity tools do not fully account for gaps when performing alignments.
Functional protein knockout of VCP is observed in a cell-based assay using stably-expressed Cas9 and Edit-R synthetic crRNA:tracrRNA in a recombinant U2OS line.
A recombinant U2OS ubiquitin-EGFP proteasome cell line (Ubi[G76V]-EGFP) was stably transduced with lentiviral particles containing Edit-R plasmid vectors expressing Cas9 nuclease and blasticidin resistance gene under the control of the indicated promoters. A population of cells with stably integrated Cas9-BlastR was selected with blasticidin-treatment for a minimum of 10 days before transfections. Cells were transfected with 50 nM Edit-R synthetic crRNA:tracrRNA complex targeting VCP, a gene required for proteasome function, using DharmaFECT 3 Transfection Reagent. After 72 hours, transfected cells were examined for EGFP+ cells (upper panel) and the relative frequency of gene editing was calculated (lower panel) based on a DNA mismatch detection assay with T7 Endonuclease I.
Knockout performance of synthetic sgRNA and crRNA:tracrRNA
A U2OS ubiquitin-EGFP cell line stably expressing Cas9 under a CAG promoter was transfected with decreasing concentrations of guide RNA (either crRNA:tracrRNA or sgRNA) targeting proteasome components PSMD7 (A) or PSMD11 (B). Transfected cells were assessed for functional gene knockout through EGFP accumulation/intensity.