Published on Aug 17, 2016
Updates!
Hi all! Here's what's been going on in the lab recently:We have constructed our base Cas9 part and are attaching various signal sequences to it via Gibson assembly. We're also expressing and purify...
Published on Jul 12, 2016
More bacteria art
Hello lovely backers! As a supporter of our project you get to see special lab notes like this one. We'll try to use this space for fun things as well as in depth videos and other media. Here are s...
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Published on Jul 08, 2016
Ordered DNA!
The fun part of gene cloning lies in the many possible ways it can go wrong (heh). We try to minimize that by simulating our experiments in silico--a pretentious way of saying on a computer. After ...
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Hi, Jim! Thank you for your question! Genetically modifying/eliminating LPS is the route we had in mind to address this problem. There is a strain of E. coli that has genetically modified LPS molecules, which do not lead to an immune response in human cells. "Endotoxin-free protein production—ClearColi™" technologyhttp://www.nature.com/nmeth/journal/v10/n9/full/nmeth.f.367.html ~ The first author of this paper is actually also the first author of the paper you're citing.
Apr 24, 2017
CRISPR Capsules: Packaging Cas9 with bacterial outer membrane vesicles
View commentYes, I think so! We like to think of this as an alternative to viral delivery and somewhat similar to lipid-mediated methods. Salient challenges would be of course managing immune response--which we can engineer as we've mentioned--and delivery efficiency (balanced out by possibly higher packaging capacity than AAVs or lentiviruses). (http://online.liebertpub.com/doi/pdfplus/10.1089/hum.2015.069)
We envision starting with topical and gut applications, so I'm not sure about cystic fibrosis. Could potentially target CFTR in intestinal cells though. We haven't seen any literature about nebulizing OMVs, so I don't think that has been done.
Jul 27, 2016
CRISPR Capsules: Packaging Cas9 with bacterial outer membrane vesicles
View commentHi Jim! Great questions!
Yes, our goal is to deliver the whole protein. This has been done for other proteins but not for Cas9 to our knowledge.
Regarding your other question, we've definitely looked into that! Previous work has shown changing the vesicles' membrane composition greatly reduces host immune response. One group working on OMVs as a cancer therapy modified lipopolysaccharides in the membrane and found the OMVs to be well-tolerated in mice. (http://pubs.acs.org/doi/abs/10.1021/nn405724x)
Jul 09, 2016
CRISPR Capsules: Packaging Cas9 with bacterial outer membrane vesicles
View commentWe will put up a video in a few days with some illustrations! Thanks for asking!
Jul 06, 2016
CRISPR Capsules: Packaging Cas9 with bacterial outer membrane vesicles
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