For sgRNA sequences, please send DNA sequences for the 18-20 nt guide RNA sequence. 18-20 nt is our recommended size though other sizes can also be synthesized. For plasmids or HDR sequences, please send the full DNA sequence desired to be synthesized in 5’ to 3’ orientation. Sequence submission forms are divided into small, 1-9 products, and large, ≥10 products. Orders of 24 or more samples will be delivered in a 96-well plate, under 24 samples will be delivered in tubes unless requested otherwise. Send all sequence submission forms to: firstname.lastname@example.org. Once submitted and approved, we will generate and send a quote for placing an order.
sgRNA and pT7sgRNA
myCRISPR sgRNA is supplied as highly purified RNA transcribed from error-free plasmid DNA made using our proprietary myDNA technology. Alternatively, we supply the error-free plasmid DNA itself containing a guide RNA sequence of your choice. In both formats, the sgRNA consists of a 5’ guide RNA sequence linked to a 3’ trans-activating CRISPR RNA (tracr) sequence that binds the Streptococcus pyogenes Cas9 nuclease. Each sgRNA contains a variable sequence of a customer defined length, 18-20 nt is recommended, and an optimized tracrRNA sequence that offers improved stability and cutting (Dang et al., Genome Biology 2015). myCRISPR sgRNA and DNA plasmid templates are delivered dry unless requested otherwise. Note that T7 transcription will add two Gs to the 5’ end of your guide RNA, unless it is designed to start with one G as part of the guide sequence. Two additional Gs at the 5’ end of the guide sequence have not been shown to affect activity (Mali et al. Nat. Biotech. 2014 and Romanienko et al. PLOS One 2016). Optionally, request a custom guide RNA sequence for binding to other nucleases, such as Cpf1, to suit a particular application.
pU6sgRNA-GFP and pU6sgRNA-GFP-Puro
Both plasmids contain a human U6 promoter for expression of your sgRNA in human or mouse cells and a CMV promoter driving GFP or GFP and puromycin expression for cell sorting and selection. The guide RNA sequence and tracrRNA sequence are designed as above for the T7 transcribed sgRNA. Each plasmid can be designed to express multiple sgRNAs by the insertion of multiple U6-sgRNA cassettes. Plasmids can be transfected with Cas9 protein/mRNA/plasmid or for transfection of a Cas9-expressing cell line. Promoters other than CMV are also available upon request. Please contact us for any modifications desired for these plasmids or to generate a completely custom sequence.
High fidelity long ssDNA and error-free plasmid templates for homology-directed repair (HDR)
We recommend long ssDNA for repair with shorter DNA sequences of 200-2000 bases and plasmids for insertion of even larger sequences up to 10 kbp in size. Submission of a DNA donor sequence, including homology arms, results in delivery of a custom DNA sequence specific for each project. Each DNA is delivered dried down and is strictly quality controlled to be RNase and DNase free and fully compatible with our other myCRISPR products. Please see the myDNA product page for a more detailed description of our synthesis capabilities.