Frequently Asked Questions

What guide RNA tool do you recommend?

We do not recommend any specific design tool, but here is a non-exhaustive list of some available tools:

Be sure your targeted sequence is followed by a PAM site of NGG and that you do not include the PAM sequence in your guide RNA!

Where can I get the Cas9 nuclease?

Depending on your application, we recommend obtaining the S. pyogenes Cas9 nuclease from NEB (Cat. No. M0641 for NLS containing Cas9), the mRNA from TriLink Biotech or for a plasmid based expression vector use Addgene, PNA Bio, or your preferred provider. Be sure the promoter for Cas9 expression works in your cell line! You can also obtain a plasmid for in vitro Cas9 mRNA synthesis from a provider of your choosing, just be sure it is for the S. pyogenes Cas9 (unless you have ordered an sgRNA that works with a CRISPR nuclease from a different species).

What is the tracr sequence used?

We use an optimized tracrRNA sequence:

5'-GUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU-3’

See Dang et al (2015), Genome Biology and Chen et al (2013), Cell for a demonstration of this improved tracrRNA sequence across multiple applications.

How should I design my guide RNA relative to T7 and U6 promoters starting transcription with a G?

If your guide sequence already starts with one or more G residues, please follow these rules: 1) for T7 transcription, two Gs will be added to the 5’ end of your guide sequence, so if your sequence starts with a G (or two), you can leave them out of your submitted sequence since at least one G will be added during transcription. 2) For U6 promoter plasmids, only one G is added to the 5’ end of your guide sequence during transcription, so if your guide sequence starts with G at the 5’ end, you can leave that first G out of your sequence.

How can I check if my sgRNA is cutting my target sequence?

For testing in vitro, several companies, such as Thermo Fisher, NEB, or PNA Bio, offer a Cas9 nuclease that can be used with your sgRNA to rapidly test cutting efficacy of your DNA sequence (usually a PCR product from your genomic DNA). This in vitro test is no guarantee your sgRNA will work in vivo, but if the in vitro reaction fails, it might be best to redesign your targeting sequence or analyze RNA quality if doing your own transcriptions. In cells, a T7 endonuclease I assay (such as NEB kit E3321S) is the fastest way to detect indels that result from cutting of your DNA target in cells, but it is not the most sensitive. You can also try the TIDE approach, which is to sequence PCR products made from primers that flank your cut site. More info on TIDE is here: https://tide-calculator.nki.nl. Prior to in vivo work, it may also be best to test your sgRNAs ability to mediate cutting on a PCR fragment in vitro.