Custom single guide RNA (sgRNA) for CRISPR-Cas9 gene editing
These guide RNAs provide a high level of gene editing functionality and are provided as transfection-ready RNA oligos. This synthetic offering gives researchers a choice in guide RNAs, when a single molecule is preferred for therapeutic applications. Synthetic sgRNAs can be designed and ordered for S. pyogenes, or any other species of Cas9 with the CRISPR Design Tool.
Edit-R CRISPR-Cas9 synthetic single guide RNAs greatly simplify the workflow of permanently knocking out genes by eliminating the time-consuming cloning of individual guide RNA expression vectors. Our chimeric sgRNAs contain both the crRNA and tracrRNA in one molecule that arrives ready-to-use and is ideal for experiments when a single molecule is preferable. Edit-R synthetic sgRNA provide the functionality and reliability of Dharmacon RNA oligos.
Single guide RNAs can be designed to target any gene using the CRISPR Design Tool. The sgRNA is synthesized using patented Dharmacon 2’-ACE chemistry with faster and more efficient coupling rates than standard RNA synthesis chemistries resulting in more full length product.
Synthetic sgRNA are column-purified, deprotected, and desalted to give highly functional, ready-to-use molecules.
For larger quantities and additional purification options for sgRNAs the Custom single-strand RNA synthesis tool can be used for ordering.
Synthetic sgRNA are compatible with any type of Cas9 nuclease, it is simply a matter of appropriate transfection conditions:
Place a custom guide RNA order, or design and order your own synthetic sgRNA, crRNA, or lentiviral sgRNA with our easy-to-use interface.
Place a custom sgRNA order for larger scales and additional purification options.
Design and order a single-strand DNA donor (≤ 150 nt) for insertion, deletion, or other alteration.
Design and order a plasmid DNA donor kit for insertion of an mKate2 or EGFP fluorescent marker, or a custom insert
Rapidly and easily assemble a plasmid donor for HDR
Quickly and efficiently build a HDR donor plasmid
PCR components for Edit-R Plasmid Donor Kits
Figure 1. Edit-R gene engineering workflow using the Edit-R Cas9 mRNA or protein co-transfected with synthetic sgRNA
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.
U2OS-Proteasome cells with integrated Cas9 (under the CAG promoter) were plated in 96-well plates at 10,000 cells per well. 24h after plating, cells were transfected with 25 nM synthetic guide RNAs (synthetic sgRNA and crRNA:tracrRNA) targeting PSMD7 and PSMD11 genes (known components of the proteasome pathway) using DharmaFECT 4 Transfection Reagent at 0.15 µg/well. After 72 hours the GFP fluorescence was measured using an Envision plate reader (Perkin Elmer). Both synthetic sgRNA and crRNA:tracrRNA have comparable gene editing functionality.
U2OS-Proteasome cells with integrated Cas9 (under the CAG promoter) were plated in 96-well plates at 10,000 cells per well. 24h after plating, cells were transfected with 6.25 - 50 nM synthetic sgRNA targeting PSMD7, PSMD11, and PPIB genes using DharmaFECT 4 Transfection Reagent at 0.15 µg/well. After 72 hours, direct cell lysis was amplified using primers surrounding the target sites and percent gene editing was estimated using a mismatch detection assay. All four concentrations of synthetic sgRNA resulted in high genomic editing for each gene target.
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.
Modifications for improved nuclease resistance: 2’-O-methyl modified nucleotides and phosphorothioate backbone linkages.
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).