We have generated and characterized a number of plasmids to express Cas9 and gRNAs in Drosophila melanogaster. They can also be used to perform genome engineering in Drosophila cell lines. All plasmids are available through the non-profit repository Addgene.
Our gRNA plasmids are compatible with a number of different protocols to deliver gRNAs for CRISPR genome engineering in the fly. Read here which protocols we recommend and why. You can find plasmid maps and cloning protocols here.
Which plasmid to use?
A particular advantage of our plasmids is that they allow expression of gRNAs with different efficiencies. Most notably, we found that the U6:3 promoter results in more efficient gRNA expression from transgenes than the U6:2 promoter, which is used in most other publicly available gRNA plasmids.Therefore, choosing the right plasmid allows you to modulate gRNA expression and thereby gene editing efficiency.
For most applications you would probably like to maximize gene editing efficiency. In this case we recommend using either plasmid pCFD3 or pCFD5, which both result in strong, ubiquitous gRNA expression. Generally, pCFD5 is more versatile, as it also allows to express multiple gRNAs simultaneously. In addition, unlike pCFD3, gRNAs expressed from pCFD5 can start with any nucleotide.
To edit the genome specifically in selected tissues, we constructed plasmid pCFD6, which can be used to express one or several gRNAs from a UAS promoter. In combination with a Gal4 driver line and a UAS-cas9 transgene, this plasmid allows conditional mutagenesis with significantly more specificity than transgenes with a conventional U6 promoter.
Expression of a single gRNA from U6:3, the strongest U6 promoter in Drosophila. Addgene plasmid 49410. For plasmid injection or transgenesis using the vermilion marker (inject into Bloomington 25709 or 25710).
Plasmid with mini-white transformation marker for easier seletion of transgenic flies.
Addgene plasmid 123366
pCFD4: U6:1-gRNA U6:3-gRNA
Expression of gRNAs from the the strong ubiquitous U6:3 promoter. gRNAs are preceeded by tRNAs and liberated by endogenous RNases. Can be used to express one or several gRNAs. We have extensively characterised pCFD5 plasmids expressing 4 gRNAs and found that gRNAs can mediate gene targeting with up to 100% efficiency regardless of their position in the array. gRNAs can start with any nucleotide. Addgene plasmid 73914
These are plasmids we have used to generate our transgenic fly lines. You might use them to generate insertions at other loci, in other fly species or to perform genome engineering in Drosophila cell lines (plasmids with act promoter only).
Addgene plasmid 62209
expresses Cas9 in flies and S2 cells