Hi everyone,
I am reading up on gene editing with CRISPR/Cas9. I wanted to understand how one assesses the gene editing efficiency of an experiment. Correct me if I am wrong, I understand the main methods used are Surveyor, digital PCR and Sanger sequencing. But then I know of some other researchers that use mainly rescue systems, like antimicrobial-resistance genes, to pick out their cell clones.
What do you guys use to quantify/assess your gene edit and why?
I would be happy to understand the field a bit more and also selection of method depending on the organism or research question.
Cheers
Felix
I'm using TIDE (https://tide.nki.nl/) to asses indel frequency from sanger seq results.
Another option we use is making NGS libraries from PCR products of the editing site.
We analyze these using Crispresso2 (https://crispresso.pinellolab.partners.org/submission).
Thanks Amr Salem .
Have you ever used regular PCR or ddPCR for screening to save some time and costs; or is Sanger seq just the go-to tool for you?
Felix Neumann I usually uses regular PCR as the first and quicker step to validate the efficiency of my guide RNA in a mixed population of cells.
1- For testing guide RNAs, I usually transfect cell for 3-5 days followed by harvesting of 80% of cells, extract genomic DNA, run regular PCR followed by T7E1 digestion
2- For generation of KO (Large deletion), I usually uses at least 2 guides, previously tested by T7E1, then co-transfect both with Cas9, and 5 days post transfection collect 80% of cells, next genomic DNA extraction, run regular PCR and sanger sequencing of the short band to confirm deletion. The rest 20% of cells will be grown for single cell clone "it takes around 4 wks"
Hopefully this help
Amr
Amr Salem Thank you very much for the detailed answer on your pre-screen with T7E1. I was exactly wondering about that step before going for sequencing - helped me a lot!
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