No gene editing in wheat: any ideas why?

[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!!

[Vmedvil]

15 hours ago, Dean22April said:

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!!

Use the longer PCR range, I think the fragment was damaged or mutated upon entry personally, also make sure your mRNA was not damaged upon entry, Cas9 will not function if it has mutations or damage in it nor will the mRNA Guide.

Both of these are most likely, DNA repair does not work that way that you said only in breaks and crosslinks etc are repaired. 

check specifically that these both match the actual protein and guide mRNA code exactly and the mRNA for the protein was translated did you put a upstream or downstream promoter on it you may want to if this keeps happening and you find the codes match, exactly for the inserted protein and the actual protein.

 

• 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

If this happens again post something, I have a different way to insert it, that will be quick and easy along with cheap that is not perfect but is more gentle way of insertion than the one that you are using.

Sticky Blunt Ends.

Though, if you are unlucky or do it wrong it will kill the plant cells used on or damage them enough that they will not grow, but this requires a high speed Centrifuge and two types of protein Endonuclease and ligase, a test tube, and a mutagen chemical along with Polyethylene Gycol 400 which I will outline if it comes to that.

Edited December 11, 2017 by Vmedvil