I am working with Xenopus and using CRISPR/Cas9 with two sgRNAs to induce a large genomic deletion (~300 kb) by injecting RNP complexes into fertilized eggs. Since these are F0 embryos, I expect mosaicism and would like to assess the deletion efficiency and mosaic rate directly in F0, without generating F1s.
For small deletions (~1 kb), I typically use PCR followed by Sanger sequencing and ICE or DECODR analysis. But for large deletions, PCR bias and low abundance of the deletion allele make this approach unreliable.
Has anyone successfully quantified large deletions (~300 kb) in F0 Xenopus embryos? Are there reliable strategies—such as ddPCR, long-range PCR combined with nanopore sequencing, or other approaches—that work well in this context?
Any advice, references, or protocol recommendations would be greatly appreciated.
Nanopore sequencing is worth trying. You should be able to see the deletion.
Note: There are/were targeted kits available to do this https://nanoporetech.com/document/targeted-amplification-free-dna-sequencing-using-crispr-cas but since they are being discontinued, so they likely don't work well. Don't know if there is a current solution.