Innovative CRISPR technology offers numerous possibilities for genome editing. Just design, deliver and detect. Sounds easy enough, right? We understand that CRISPR editing can be daunting so here are our top 10 tips for a successful CRISPR edit.
- Know what edit you want to make
What’s your goal: to knockout a protein or make a specific change? The type of edit defines experimental design and how you deliver the editing tools.
- Find your PAM sites
Next, find the potential PAM sites close to your edit site. For Sp. Cas 9 protein, the PAM site is NGG and it cleaves three bases 5’ of the PAM site. For a protein knockout, the edit site can be in the first few exons. For a specific change via HDR, the cut site closest to the edit site increases efficiency.
- Design your guide RNA
We’ve tried to make designing your guide RNA (gRNA) easier. Our TrueGuide Synthetic gRNA is made of the crRNA (18-20 bases 5’ of the PAM site) and a tracrRNA.
- Specific changes need an HDR template
A good HDR template has the precise change you want to make and homology arms. For a successful edit, the template needs to be present when the cleaved target site is being repaired.
- Choose the most efficient Cas9 format
There are so many formats of Cas9 protein – which do you choose? Our TrueCut Cas9 Protein v2 is the most efficient format. Learn more about the efficiency of Cas9 here.
- Test multiple guide RNAs
gRNAs can have different cleavage efficiencies so try to test at least three in order to find the most efficient one.
- Know your cells
The idea is you want to deliver the editing tools with the highest transfection efficiency so you must know your cells. The better the transfection efficiency, the better the editing efficiency. Choose the method that gives the highest delivery efficiency based on your cell type. Check out our free validated protocols for a wide range of cell types here.
- Include a positive control
Try to include transfection controls to monitor cell health and reagent function.Use a positive control gRNA with known cleavage efficiency. Check out our ready-to-use controls: TrueGuide Synthetic gRNA Controls
- Confirm the gene editing efficiency
Test the editing efficiency of multiple gRNAs using a quick method like the Genomic Cleavage Detection kit.
10. Use the most efficient gRNA in your experiment
Now that you know your editing efficiency, you can transfect the most efficient gRNA into your cell using the optimized transfection conditions to achieve highest editing efficiency.
Are you looking to get started with CRISPR?
Join our Hands-on CRISPR training course our experienced team has designed a comprehensive four-day CRISPR workshop comprised of both lectures and hands-on laboratory work at our state-of-the-art training facility. To learn more visit thermofisher.com/crisprworkshop
Have you mastered the art of CRISPR editing? Share your tips and tricks in the comment box below.
For Research Use Only. Not for use in diagnostic procedures.
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