Plasmids

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.

 

gRNA plasmids

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.

 


pCFD1: U6:1-gRNA

Plasmid for ubiquitous expression of a single gRNA. Results in intermediate expression level. Available from Addgene (Plasmid 49408). For plasmid injection or transgenesis using the vermilion marker (inject into Bloomington 25709 or 25710).

paper
cloning protocol
plasmid map

CFD1

pCFD2: U6:2-gRNA

Plasmid for ubiquitous expression of a single gRNA. Results in the lowest expression level. Available from Addgene (Plasmid 49409). For plasmid injection or transgenesis using the vermilion marker (inject into Bloomington 25709 or 25710).

paper
cloning protocol
plasmid map

CFD2

pCFD3: U6:3-gRNA

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).
paper
cloning protocol
plasmid map

CFD3

pCFD4: U6:1-gRNA U6:3-gRNA

Tandem expression vector. For most applications pCFD5 will be the better choice. Addgene plasmid 49411

cloning protocol
plasmid map

CFD4

pCFD5: U6:3-t::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

paper
cloning protocol

pCFD5

pCFD6: UAS-t::gRNA

Plasmid for tissue-specific gRNA expression with the Gal4/UAS system. Can express one or multiple tRNA-flanked gRNAs. We use this plasmid to create large-scale gRNA libraries for functional genomic screening in Drosophila (see here).

paper
pCFD6cloningprotocol


Cas9 plasmids

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).

pAct-cas9

Addgene plasmid 62209
ubiquitous expression
highly active
expresses Cas9 in flies and S2 cells

actCas9plasmids

pNos-cas9

Addgene plasmid 62208
germline specific expression
used in fly line CFD2

nosCas9plasmids

pAct-Fok1:dCas9

Addgene plasmid 62211
ubiquitous high expression
high specificity
activity validated in vivo

actFokCas9plasmids

pNos-Fok1:dCas9

Addgene plasmid 62210
germline specific expression
high specificity
activity validated in vivo

nosFokCas9plasmids