For a general overview of this project I refer you to my previous tweets about the library and the associated toolbox:https://twitter.com/crisprflydesign/status/1128015203629699072 …
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For a general overview of this project I refer you to my previous tweets about the library and the associated toolbox:https://twitter.com/crisprflydesign/status/1128015203629699072 …
In the revised version of the preprint we have added many more fly lines and plasmids, have revised text and figures to be more concise and have added two main innovations. Let's look at the new things...
First, we have updated our sgRNA vector pCFD6 with FRT sites either side of the sgRNA cassette (now pCFD6.FRT). These can be used to exchange sequences before or after the sgRNAs in vivo.pic.twitter.com/KXVKWa3Voo
For example, you can swap the UAS-DSCP promoter in our lines with a promoter such a U6:3. Our default is UAS, since we have shown previously that this drastically reduces leaky mutagenesis that arises independent of Gal4. But with weak Gal4s...
... sgRNA expression can become limiting. In these cases you might prefer U6:3, which is very strong and ubiquitous. With pCFD6.FRT transgenes you can efficiently swap the promoter:pic.twitter.com/raOUqZagpp
Another possible application of these FRT sites is to add additional elements behind the sgRNAs. For example additional sgRNAs. This could be used to make a gene knock-out more penetrant or to target several genes in parallel.pic.twitter.com/teAjFOQVbm
This is now described in Figure 3 and we already have 100s of plasmids using pCFD6.FRT and many fly lines are becoming available.
The second innovation is an improved method to map the spatial mutagenesis pattern of any Gal4 UAS-uCas9 fly line. Since CRISPR creates permanent mutations and requires only minute amounts of Cas9, these patterns often diverge from the expected.
For example, in the third instar wing imaginal disc ptc-Gal4 is a popular driver for perturbations, as it expresses in a very distinct stripe along the AP-boundary. Here visualized by driving UAS-GFP.pic.twitter.com/KgkE38z3A7
But if you use ptc-Gal4 for CRISPR mutagenesis, you end up with mutations not only in the stripe, but throughout the anterior half. This likely reflects broader expression in early development or low expression in these cells.pic.twitter.com/iOxvz48YlL
This can also be observed with other Gal4 lines. So validating the spatial mutagenesis pattern when starting to use a new Gal4 UAS-uCas9 line is generally a good idea. Previously we did this by CRISPRing a gene and then stain for the protein.
But this is not a very practical approach. Fortunately, in the meantime others have developed reporters to visualize Cas9 mediated mutations and these can be used for mapping mutagenesis patterns.
We are using CIGAR, developed by Erich Brunner and Ryohei Yagi in Koni Basler's lab, which turns CRISPR edits into GFP expression. Shout out to Erich for openly sharing tools prior publication! Their paper is out in the meantime:https://www.life-science-alliance.org/content/2/3/e201800267 …
So now you can use CIGAR or a similar reporter (e.g. Tzumin Lee's lab has publs something similar) to light up all cells in the animal that are subject to mutagenesis.
Patterns marked by fluorescence are not entirely robust, as no reporter to-date can mark 100% of CRISPR edits (large deletions being main problem). So it is best to average several samples. We use image registration to produce overlays (right panel).pic.twitter.com/vCdYsXslVf
In summary, we believe that the new additions make our resource much more versatile (through the use of pCFD6.FRT) and highlights strategies to characterize conditional CRISPR mutagenesis experiments in vivo (e.g. map spatial patterns).
Puh, this got a bit long. Congratulation if you made it this far! We are always happy to receive feedback. Many thanks for reading. Bye.
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