Question: How to confirm correct integration of a reporter using the CRISPR/Cas9 mediated homology-repair DNA pathway?
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gravatar for Andrea
3.7 years ago by
Andrea10
Andrea10 wrote:

Dear Biostars Community,

Please consider the following procedure:

Human embryonic stem (hES) cells were nucleofected with a literature-confirmed CRISPR and a reporter with flanking homologous arms. The reporter carries GFP and Puromycin which were used to select and expand the clones.

To evaluate that the reporter integrated in the correct site, I used 5′/3′ junction PCR on the left and right integration sites. Junction PCR meaning that the primers span from genomic to reporter boundaries which allows to confirm site-specific insertion of the reporter.

This worked in my control cells, pooled HEKs transfected with the same CRISPR and reporter. However, in my hES clones (which are still GFP positive after three passages of clonal expansion, meaning that the reporter must have stably integrated somewhere) the PCR did not work.

Now I am thinking how to troubleshoot this issue. I will send the control-amplicon for sequencing to check whether my PCR product is reliable.

But I am wondering how could I be able to find a random integration site? I could send some extracted gDNA with a primer form the 3’/5’ end of the reporter and sequence into the genomic sequence, but I don’t think normal Sanger sequencing will be sufficient for this purpose.

With that, any suggestions on how to find the locus where a reporter might have integrated would be greatly appreciated. Alternative, are there any other methods to check site-specific integration then using 5′/3′ junction PCR?

Thanks very much!

Andrea

sequencing crispr • 1.5k views
ADD COMMENTlink written 3.7 years ago by Andrea10
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