What direction should we pursue for CRISPR/Cas9 in wheat?

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What direction should we pursue for CRISPR/Cas9 in wheat?

Postby Dean22April » Oct 26 2017 4:48 pm

So I am in a group testing whether the plants we have of a certain wheat line are expressing the Cas9 transgene and if the gene of interest that we've tried to target has been edited by CRISPR/Cas9.

So far I have: -Extracted DNA and run a PCR and gel electrophoresis to confirm some (around 75%) of the plants have at least the starting fragment of the Cas9 gene
-Extracted RNA and run an RT-PCR and gel electrophoresis to confirm some of those have the mRNA for the Cas9 (so are expressing the DNA) - around half were
-Extracted and run DNA for the guide RNA (which all showed to be expressing worryingly)
-Quantified the amount and quality of our mRNA using a bioanalyser
-Sequenced the guide RNA and some of the Cas9 DNA samples (which we'll get the data for tomorrow).

So now we only have 3 weeks left in the lab and we need to decide what to do, whether we go back and re-do some processes or check for expression of proteins or editing of the gene. We are definitely planning to test whether the gene itself has been edited, but don't know what else would be useful. Any ideas what direction we should pursue?
Thanks!!
Dean22April
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Re: What direction should we pursue for CRISPR/Cas9 in wheat

Postby 29yrsExperience » Nov 02 2017 5:26 pm

Hi Dean22! I have no experience with CRISPR per se, though I have built targeting vectors-the older way of gene targeting. It seems to me you need to look for the protein you targeted, and see if the desired alteration took place. What kind of edit were you trying to make (partial deletion, insertion, knock-out)? Think about how you would identify the altered protein (is it bigger, smaller, absent?) on a PAGE gel for instance. Got an antibody for it? Also, depending on the kind of edit you could design PCR primers to distinguish between edited sequence and the unedited genomic sequence in your controls. Hope you have made some progress; good luck with the next two weeks!
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Re: What direction should we pursue for CRISPR/Cas9 in wheat

Postby Dean22April » Nov 27 2017 5:23 pm

29yrsExperience wrote:Hi Dean22! I have no experience with CRISPR per se, though I have built targeting vectors-the older way of gene targeting. It seems to me you need to look for the protein you targeted, and see if the desired alteration took place. What kind of edit were you trying to make (partial deletion, insertion, knock-out)? Think about how you would identify the altered protein (is it bigger, smaller, absent?) on a PAGE gel for instance. Got an antibody for it? Also, depending on the kind of edit you could design PCR primers to distinguish between edited sequence and the unedited genomic sequence in your controls. Hope you have made some progress; good luck with the next two weeks!


Hi thanks for getting back to me and sorry for my delay! We were looking to edit via indels, thanks so much for this, we've been extended another two weeks (after our NGS took a little longer than expected) so we're now going to check for edits when we get the data there then design specific PCR primers around any edit sites
Dean22April
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