Zebrafish Knockins

I was wondering if anybody had experience with successful zebrafish knockins using CRISPR. I am trying to knockin a promoter+RFP at a particular location. Following injections I see the RFP in the correct location and I pick out the positive embryos. However, when I try to screen individual F0 embryos using PCR (one primer on CFP and one outside the 800bp homology arm) to see whether I am getting any HDR I don't get any amplification. I am trying to decide if something is wrong with my Cas9/sgRNA injections or whether the PCR isn't working (I've tried PCR gradients and multiple extraction protocols). I was wondering if anybody has any ideas as to how I can confirm at least some fragments are getting integrated in my embryos before I go to all the effort of screening them for founders. The only thing I can think of is pooling large quantities of embryos and I don't have many (maybe 20+ positives) each time.


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Pink Hydraover 4 years ago

have you tried to use primers for actin p.e. to discard the PCR-not-working possibility?

I do this often in flies as an internal control.

Good luck!

Tan Faunover 4 years ago

Yes, sorry. My DNA extracts appear fine for other primers as I have tested for that.

Cyan Yetialmost 4 years ago

I wonder if you solved your problems with your knock-in in zebrafish. I have the same problem, but when I try to screen individual F0 embryos extracting gDNA and performing an amplification by PCR (two primers flanking the region containing the ssDNA template) followed by sequencig to see whether I am getting any HDR I don't get any base change. Could you tell me were is your concentrations of gRNA + Cas9 + ssDNA in injections?

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