To successfully edit your genome of interest, one critical step is to test the sgRNA you have designed. Fortunately there are programs that have been developed such as CRISPRscan for zebrafish, SSC, Sequence Scan for CRISPR, or WU-CRISPR that you can use to predict the efficiency and the suitability of the sgRNA. However, the prediction efficiency of these computational tools are far from perfect. While most of the time your sgRNA is likely to work, sometimes it won’t. Therefore, you need to test your sgRNA and the cleavage efficiency of your Cas9 using a reliable system.
There are easy ways to test your sgRNA prior to using it in your favorite system. These methods are in-vivo or in-vitro based approach.
In-vitro sgRNA Testing
The in vitro method for testing sgRNA is popular and easy to establish and use. To begin, synthesize the sgRNA using T7 in-vitro transcription and gel purify. You can start with two annealed single stranded oligonucleotides or a plasmid clone as your template. There are a few methods published and protocols available online on the sgRNA synthesis.
Next, add your sgRNA to your DNA template (often a PCR product of your genomic region of interest), your Cas9 mRNA or protein and incubate the mix at 37°C. Make sure you run your controls! For a negative control, I include a tube that doesn’t contain the Cas9. In parallel, I run the reaction with a proven sgRNA for a positive control. Incubate for 1-2 hours at 37°C, and then run the samples on a 2% gel. If you see two or more bands, congratulations! You have successful cleavage of your DNA template.
This testing is very easy to perform and it is easy to tease out whether things are working within few hours. There are even commercial kits to simplify the task. Beware though, the system is imperfect. Some sgRNAs that are efficient in this assay do not work well in-vivo system. This is usually due to the cellular environment preventing the formation of the Cas9/sgRNA complex and/or to inhibiting cleavage.
In-vivo sgRNA Testing in Cell Lines or Embryos
In-vivo testing systems are certainly more reliable for testing your sgRNA or Cas9 enzyme. However, they are a little bit time consuming as they require a cloning step; you definitely won’t get your results in a few hours.
There are many ways to test your sgRNA in-vivo. You can either test thee guides in a cell line in tissue culture or in a model organism. For model organisms, you test your sgRNA directly in embryos then culture the embryo to a stage in which you can extract the DNA for genotyping and T7 endonuclease assay (T7E1).
In Cell Lines
For testing in a cell line, you deliver your sgRNA and Cas9 in to the cells using your favorite transfection method. A good way to do this is to clone your sgRNA into a vector that also contains the Cas9 gene, a U6 promoter, and a GFP reporter (For instance use the Px458 vector from Zheng’s lab, available on Addgene). After transfection, check the level of fluorescence in your cell to find the transfected cells, then grow the cells and extract the DNA (you don’t need to go clonal, the only things you wish to assess is the efficiency of your sgRNA). Use PCR to check the editing using a T7E1 assay.
There are many variants to this system in cell lines. For instance, instead of GFP, you can select for transfected cells using an antibiotic resistance cassette in the plasmid (Px459 from Zheng’s lab on a puromycin resistance cassette, also available on Addgene).
In Model Organisms
The principle for testing in a model organism is the same as described for cell lines. The best way to test your CRISPR/Cas9 system and your sgRNA is to deliver your complex sgRNA and Cas9 into the embryos by electroporation or microinjection. You don’t necessarily have to use a plasmid vector. For instance, in mouse embryos you don’t need to clone your sgRNA into a plasmid system. You can either use the Cas9 mRNA or protein with your sgRNA. After Cas9 and sgRNA delivery, culture your embryos to a stage in which you have enough DNA yield for extraction. In mice it is usually 5 days of culture to blastocyst stage. Extract the DNA from your embryos then test using the T7E1 endonuclease assay. Alternatively, you can use Sanger sequencing.
Testing a sgRNA for its efficiency is relatively easy to implement in your laboratory. While online prediction tools are improving, practical testing of your sgRNA guarantees the best chance of success for your editing.Image Credit: Duncan Hull