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CRISPR-Cas9 genome editing with a synthetic 99-mer single guide RNA
Figure 2. 2'-ACE chemistry was used to synthesize a 99-mer sgRNA (Hsu et al. 2013, Briner et al. 2014) targeting PPIB, which was then purified by HPLC. A U2OS cell line stably expressing Cas9 nuclease from the CAG promoter was plated at 10,000 cells per well in 96-well format one day prior to transfection. sgRNA (25 nM) or synthetic crRNA:tracrRNA (25 nM) was transfected into duplicate wells using DharmaFECT™ 3 transfection reagent (0.25 μL/well). After 72 hours, direct cell lysis was amplified using primers surrounding the target site on the PPIB gene and gene editing efficiency estimated using a mismatch detection assay (Edit-R™ Synthetic crRNA Positive Controls - Protocol). The 99-mer synthetic sgRNA for target gene editing resulted in high efficiency indel formation (data shown are from two duplicate experiments; A), and high editing efficiency was also achieved for synthetic crRNA:tracrRNA (B).
The CRISPR-Cas9 system allows researchers to quickly edit genes for functional protein knockout in mammalian, fish and plant genomes, among others, and consequently has dramatically transformed biological research. The natural CRISPR-Cas9 system requires three components: 1. Cas9 nuclease, 2. CRISPR RNA (crRNA) comprised of spacer-derived sequence and of repeat-derived sequence, and 3. tracrRNA, which hybridizes to the crRNA through repeat-derived sequences. The crRNA:tracrRNA complex recruits the Cas9 nuclease and cleaves DNA upstream to a protospacer-adjacent motif (PAM). The crRNA and tracrRNA can be linked together with a loop sequence for generation of a chimeric single guide RNA (sgRNA; Hsu et al. 2013). sgRNA can be generated for DNA-based expression or by chemical synthesis. With traditional chemistries, such as 2'-silyl (TBDMS or TOM) protection strategies, it can be challenging to accurately and efficiently synthesize RNA greater than ~ 70 bases. Using patented Dharmacon 2'-ACE chemistry (Scaringe et al. 1998, Scaringe et al. 2004), long RNA can be routinely synthesized with faster coupling rates, higher yields and greater purity and is ideal for generating synthetic sgRNAs.
Although a natural synthetic two RNA (crRNA:tracrRNA) system is very efficient and cost-effective for most applications, researchers working with in vivo and ex vivo models have indicated a preference for a sgRNA system. The advantages to using a synthetic sgRNA compared to plasmid-expressed or in vitro transcribed (IVT) sgRNA include:
Below is one example of how to design a sgRNA (Hsu et al. 2013) for chemical synthesis:
Custom single-strand RNA synthesis ordering supports lengths up to 105 nt at the 0.4 µmol synthesis scale and is suitable for ordering single guide RNAs. It is recommended to include HPLC purification and 2’-deprotect/desalt to reduce the presence of non-full length RNAs in the final product. Final amounts typically achieved with this processing are 3-5 nmol but will vary depending on the RNA length.
Design and order your synthetic sgRNA
Optimized tools for high-confidence genome engineering
Configure the optimal promoter for your cell type to ensure robust Cas9 expression or explore DNA-free options
Proper controls are essential to assessment of CRISPR-Cas9 genomic editing experiments