I PCRed one of the native CRISPRs out, believe it had AGAAW or something like that as the PAM. I didn't have enough funding or time to fully clone it, but I do plan on it soon. If I remember correctly, the crRNA is on the 3' of the Cas9 rather than the 5' like normal Cas9. Would be convenient if you need hard regulation of it's expression.
-- https://www.amazon.com/gp/product/B001MV5PB0/ref=oh_aui_search_detailpage?ie=UTF8&psc=1
PCRed a 500bp fragment of that Cas9 off of this. It was a couple years ago though. There's like 2-3 CRISPRs in it I think, depending on what strain you have.
I also think I have a broad range gram positive plasmid that expressed GFP from B subtilis. Would have to dig that up. Would most likely work in S thermo. I think the yogurt starter has one other strain though.
-Koeng
On Tuesday, May 31, 2016 at 5:38:11 AM UTC, Patrik D'haeseleer wrote:
On Tuesday, May 31, 2016 at 5:38:11 AM UTC, Patrik D'haeseleer wrote:
Here's an interesting thought I've been toying with:One of the classical yogurt making bacteria is Streptococcus thermophilus, a close relative of S. pyogenes, which is the source of the canonical Cas9 gene.Turns out that S. thermophilus has a native Cas9 system. So in theory, all we need to do genome editing in S. thermophilus would be to provide it with an artificial CRISPR casette!Completely bypasses any IP issues around Cas9 as well... :-)Patrik
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