How do you screen for precise editing?
At the Allen Institute we use droplet digital PCR (ddPCR) to determine how many copies of GFP our clonal cell lines have (hopefully 1 or 2!) and to determine if there is any plasmid backbone incorporated into the clone’s genome (hopefully not, but this happens frequently!). This is high throughput and the assay never changes, regardless of the gene tagged.
Clones that have 1 or 2 copies of GFP and no plasmid backbone by ddPCR are then subjected to a series of gene-specific junctional PCR assays where 1 primer is in GFP and 1 primer is outside of the homology arm region to make sure the GFP is in-frame, and on-target. Even in clones that pass our ddPCR analysis, we find evidence of mis-repair during HDR through these junctional assays- usually in the form of a PCR product that is too big, suggesting a partial duplication or partial insertion of plasmid backbone. This is also high-throughput and requires relatively little primer optimization for each gene. We run the PCR products on a Fragment Analyzer which saves time and effort compared to running gels.
We also amplify the full allele and Sanger sequence all of our PCR products (tagged allele and untagged allele in heterozygous clones). Sometimes we’ll find evidence of NHEJ on the untagged allele of heterozygous clones, which we don’t want. Sometimes we identify small insertions in the tagged allele that were too small to be picked up simply by looking at the size of the PCR product, so Sanger sequencing is a must for us!
What do you do in your lab?
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