Fluorescent Tagging ssODN for HDR repair template

[Josh]

Hi, co-op student here,

We are trying to knock out a point mutation in our hard to transfect ovarian cancer cells and repair it with the wild type repair template.

Following the protocol outlined in Nature paper Ran et al 2013: Genome Engineering using the CRISPR-Cas9 system, we are using PX458 as our Cas9 + sgRNA plasmid, and a ssODN repair template that we are co-transfecting via electroporation.

We can observe the PX458 successful entry into the cells due to the GFP marker built in the plasmid, but I was wondering if there would be a way to screen for the ssODN repair template (i.e either by fluorescence or other means) without having to grow mass amounts single cells and sequence them after the fact .

Any thoughts on this would be great.

Thanks,

Josh

[CB]

I am assuming that your repair template has a slightly different sequence than the parental cell? Maybe ss oligo has silent mutations to destroy the PAM site or guide RNA binding? If so, you can design a F or R primer specific to the repair template. PCR amplify your clones and the positives should be the ones with the mutation. You can enrich for those that have been transfected by GFP sorting positive cells into a 96 well plate to get one cell per well. Grow these up, make a replica plate, lyse the cells with quickextract and run the pcr. Grow up the positive clones and validate those with sequencing, etc. 

[Carter]

Hi Josh, 

Which ovarian cancer cell line are you trying to transfect? Did you ever think about adding a fluorophore such as Cy3? I am not sure if it will impact HDR?

Regards, 

Carter

On Wednesday, June 1, 2016 at 6:36:08 PM UTC-5, Josh wrote: