Why Didn't We Find Any Gene Editing In Our Wheat Plants?

[Dean22April]

We were wondering why we didn’t have any edits in our wheat plants using CRISPR/Cas9 technology and what we could possibly do to ensure we would have edits in the future?

So, our experiment consisted of:

1. 40 plants that had been successfully transformed using biolisitcs with a Cas9 construct and sgRNA
2. Of these we genotyped to find out which had Cas9 DNA, and of these which were expressing Cas9 mRNA, we used PCR to determine this
3. We then worked out which of the 40 plants had the sgRNA
4. The plants that had both sgRNA and Cas9 we took forward and sequenced using Next Generation Sequencing to determine if the CRISPR/Cas9 construct had performed any edits (insertions or deletions)

We found no editing in any of the plants, but unsure why this would be the case and what we could do to ensure editing in the future?

Ideas I’ve identified so far:

• The Cas9 mRNA wasn’t translated properly: we’d test this doing a western blot?
• The wheat genome is heavy in repeat regions and has three genomes (hexaploid) so the DNA was repaired more easily therefore any edits were simply repaired
• When checking for Cas9 fragment not the entire Cas9 gene had been inserted: in future we’d test for entire gene using long range PCR

Any help or ideas would be hugely appreciated, a bit frustrating we didn't find any edits so now trying to work out why, and what we could do to ensure we'd get some in the future.

Thank You!!