Naked Science Forum

Life Sciences => Cells, Microbes & Viruses => Topic started by: Dean22April on 10/12/2017 12:39:49

Title: Why didn't I find gene editing in my wheat plants?
Post by: Dean22April on 10/12/2017 12:39:49
(Not sure if this is the right forum but unsure where else to post) Hi, I'm in a team investigating CRISPR/Cas9 editing in wheat. We were wondering why we didnt 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:


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 Ive identified so far:


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!!
Title: Re: Why didn't I find gene editing in my wheat plants?
Post by: chris on 15/12/2017 23:27:43
A few ideas spring to mind:

1) Maybe the edited cells were no-viable and were lost to follow up?

2) Maybe the strand-breaks introduced by CRISPR were repaired using a complementary sequence from the other parallel genome, as you surmise, rather than the guide sequence?

 - there is a precedent for a strange thing like 2) happening:

When scientists "fixed" human embryos carrying a defect in a gene linked to a form of developmental heart condition, they found that the error was fixed from the complementary region of the corresponding healthy chromosome, rather than the supplied patch. Might this happening in your plants?
https://www.thenakedscientists.com/articles/science-news/gene-editing-corrects-embryonic-heart-defect
Title: Re: Why didn't I find gene editing in my wheat plants?
Post by: Dean22April on 17/12/2017 16:48:39
Hi, thanks for your reply!!

I'm a little confused, what do you mean by 'lost to follow up'?

Thanks for sending that article as well that is very interesting and I hadn't come across that at all, the only worrying thing is that our CRISPR/Cas9 constructs should only have edited using NHEJ which is itself a 'repairing' mechanism, all be it a faulty one, not HDR. Or was that not what you mean?

Thanks again! :D

A few ideas spring to mind:

1) Maybe the edited cells were no-viable and were lost to follow up?

2) Maybe the strand-breaks introduced by CRISPR were repaired using a complementary sequence from the other parallel genome, as you surmise, rather than the guide sequence?

 - there is a precedent for a strange thing like 2) happening:

When scientists "fixed" human embryos carrying a defect in a gene linked to a form of developmental heart condition, they found that the error was fixed from the complementary region of the corresponding healthy chromosome, rather than the supplied patch. Might this happening in your plants?

Title: Re: Why didn't I find gene editing in my wheat plants?
Post by: Dean22April on 17/12/2017 17:04:03
Okay I've just re-read the article and missed the first time that CRISPR ignored the patch they'd put in, so this may have happened in our plants

Hi, thanks for your reply!!

I'm a little confused, what do you mean by 'lost to follow up'?

Thanks for sending that article as well that is very interesting and I hadn't come across that at all, the only worrying thing is that our CRISPR/Cas9 constructs should only have edited using NHEJ which is itself a 'repairing' mechanism, all be it a faulty one, not HDR. Or was that not what you mean?

Thanks again! :D

A few ideas spring to mind:

1) Maybe the edited cells were no-viable and were lost to follow up?

2) Maybe the strand-breaks introduced by CRISPR were repaired using a complementary sequence from the other parallel genome, as you surmise, rather than the guide sequence?

 - there is a precedent for a strange thing like 2) happening:

When scientists "fixed" human embryos carrying a defect in a gene linked to a form of developmental heart condition, they found that the error was fixed from the complementary region of the corresponding healthy chromosome, rather than the supplied patch. Might this happening in your plants?