Hi everyone!
I've used clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) gene editing approach to knock out one gene. The knock down was performed via lentiviral transduction of CRISPR components.
It looks beautifully at the protein level and qPCR levels.
On the western the first 2 lanes are either with WT or EV. And then 3 with KO (3 different concentration of the virus). But when I've checked the sequence I don't see any differences with the WT: 100% the same in the region which was amplified.
This is all "bulk". I can't explain it. Please help.
Hi Amr!
I've sequenced PCR product of a region of interest from genomic DNA. In the sequence I see the gRNA with PAM very good.
Hello Maria Simanovich
I suggest sequencing the WT and interested target region and then use any software (e.g. synthego) to analyse the editing. By looking the sequence data it seems that there is editing (may be Biallelic). Editing generally appears 3-4 base pair upstream of PAM sequence in case of Cas9.
Best of luck.
It is possible for a CRISPR/Cas9-mediated gene knockout to result in a protein and qPCR confirmation without detectable changes in the DNA sequence. This could be due to the editing resulting in changes outside of the sequenced region or biallelic editing, where both the edited and unedited alleles are present in the same sample. One suggestion from a fellow researcher is to sequence both the wildtype and edited target regions and use analysis software to confirm editing. Another recommendation is to ensure that the primers used for PCR amplification flank the targeted region of the spacer to increase the chances of detecting editing.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
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