Edit-R CRISPR-Cas9 Gene Engineering Platform

The Dharmacon Edit-R Gene Engineering platform is based on the Type II CRISPR-Cas9 system from the bacteria Streptococcus pyogenes which can be engineered and adapted to edit genes in mammalian cells. When Cas9, the endonuclease component of a CRISPR-Cas system, is complexed with two RNAs called the CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA), it forms a complex that cleaves DNA. This flexible system can be exploited to induce site-specific genome modifications to program, regulate and precisely interrogate gene function.

The Dharmacon Edit-R CRISPR-Cas9 platform includes the three critical components required for gene editing in mammalian cells, based on the natural S. pyogenes system:

• A protein, mRNA, or lentiviral vector expressing a mammalian codon-optimized gene sequence encoding Cas9 nuclease
• A chemically synthesized trans-activating CRISPR RNA (tracrRNA), and
• A chemically synthesized CRISPR RNA (crRNA) designed to the gene target site of interest

We guarantee that these guides will provide successful editing at the target site when delivered as described in the Technical Manual.

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.

High percentage of gene editing using the T7E1 assay to determine indel formation of the Edit-R AAVS1 cutting controls

Jurkat cells (250,000/96w) were nucleofected with (SE buffer, program CL-120) 50 pmol of RNP (40 µM NLS-Cas9 + 80 µM sgRNA) in triplicate. U2OS cells hEF1α-Cas9 were transfected using 0.15 µL Dharmafect 4/96w + 50 µM sgRNA (incubated at room temperature for 20 minutes in MEM solution).

After 72 hours cells were harvested for direct lysis (+ Rnase + proteinase K). Primers per spacer region were used to amplify 450-800 bp genomic amplicon from the edited and unedited samples. A hot-start Phusion II touch-down PCR program was used to generate amplicons for each sample (Tm ~65 for each). PCRs were directly incubated with T7 endonuclease I and ran on a 2% agarose SyberSafe gel @ 80V for 90 minutes.

TIDE analysis confirms high efficiency of indel formation using Edit-R AAVS1 cutting control guides

Jurkat cells (250,000/96w) were nucleofected with 50 pmol of RNP (40 µM NLS-Cas9 + 80 µM sgRNA) in SE buffer and program CL-120, in triplicate. U2OS cells were transfected using 0.15 µL Dharmafect 4/96w + 50 µM sgRNA (incubated at room temperature for 20 minutes in MEM solution).

After 72 hours cells were harvested for direct lysis. Primers per spacer region were used to amplify 450-800 bp genomic amplicon from the edited and unedited samples. A hot-start Phusion II touch-down PCR program was used to generate amplicons for each sample. PCRs were sent for Sanger sequencing. Sequencing files were analyzed by tracking of indels by decomposition (TIDE).

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, M.L., 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