Transgenes are a popular way to supply Cas9 for Drosophila genome engineering. They provide high efficiency and make gene targeting more robust, as well as giving you the opportunity to restrict CRISPR/Cas activity in time and space. Below you find details of the cas9 transgenic Drosophila melanogaster lines we and our contributors have generated to date. These are available from the Bloomington Drosophila Stock Center (here) or can be directly requested from the source lab. Please see our pre-print for a systematic characterization of these lines and how they compare with some of the published cas9 stocks generated by others.

Ubiquitous activity

name

act-cas9

description

This fly line has strong ubiquitous expression of Cas9. We used the human codon optimised cas9 from the Church lab (Addgene plasmid 41815) for this line. The coding sequence was PCR amplified and cloned behind a 4.4kb fragment of the upstream regulatory sequence of the act5C gene. The resulting plasmid was integrated on attP2A on the X chromosome (Bloomington 24480). Early shipments of this line had a forked allele floating. Furthermore, there is a paralysis/seizure phenotype floating, that is apparent upon taping the tubes on the table. This phenotype is already present in the parental attP2A stock. Nadine Muschalik has now produced a clean stock without these floating alleles.

genotype

y1 P(act5c-cas9, w+) M(3xP3-RFP.attP)ZH-2A w*

activity

The act-cas9 line has been tested and successfully used to mutagenise several genes in a number of laboratories. It is our most active line with very high activity in soma and germ line. Crossed to transgenic gRNAs with high activity it closely mimics the mutant phenotype of the target gene. In combination with transgenic or injected gRNAs it is ideal to generate novel alleles in non-essential genes.

stock ID

Internal: CFD1; Bloomington stock center: 54590; VDRC: 300000

reference

Port F, Chen HM, Lee T, Bullock SL.: Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):E2967-76.

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Germ line restricted activity

name nos-cas9 description This line uses again the untagged human codon optimised cas9 from the Church lab (Addgene 41815). The open reading frame was cloned into a plasmid containing the nanos promoter and 3’UTR to give germ line specific expression. The plasmid was based on the one used to generate the popular nos-PhiC31 integrase line (a gift from Johannes Bischof (Basler lab, Zurich)). The transgene is inserted at attP2A (Bloomington 24480) on the X chromosome. There is a paralysis/seizure phenotype floating, that is apparent upon taping the tubes on the table. This phenotype is already present in the parental attP2A stock. genotype y1 P(nos-cas9, w+) M(3xP3-RFP.attP)ZH-2A w*

activity

This line has intermediate, germline restricted activity. In combination with a gRNA transgene it is ideal to generate lethal mutations. It is the line we use most in our lab.

stock ID

Internal: CFD2; Bloomington stock center: 54591; VDRC: 300001

reference

Port F, Chen HM, Lee T, Bullock SL.: Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):E2967-76.

name nos-Gal4VP14 UAS-cas9 description Here we combined the popular nanos-Gal4VP16 driver with one of our UAS-cas9 (CFD3, see below) constructs. Both transgenes are located on the third chromosome genotype P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC]=UAS-Cas9.P}attP2 P{w[+mC]=GAL4::VP16-nos.UTR}CG6325[MVD1]

activity

The line has high activity, predominantly in the germ line, but also significant somatic activity. For most experiments lines CFD1 and CFD2 will be better suited.

stock ID

Internal: CFD3_nos; Bloomington stock center: 54593; VDRC: 300003

reference

Port F, Chen HM, Lee T, Bullock SL.: Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):E2967-76.

name nos-cas9:GFP description This line uses a human codon optimised cas9 from the Doudna lab (Addgene 42234). The open reading frame was cloned into a plasmid containing the nanos promoter and 3’UTR, based on the one used to generate the popular nos-PhiC31 integrase line (a gift from Johannes Bischof (Basler lab, Zurich)). The transgene is inserted at attP2A (Bloomington 24480) on the X chromosome. genotype y1 P(nos-cas9:GFP, w+) M(3xP3-RFP.attP)ZH-2A w*

activity

The line has high activity, predominantly in the germ line, but also significant somatic activity. For most experiments lines CFD1 and CFD2 will be better suited. It shows nice GFP fluoresence in the nuclei of the female germ line.

stock ID

Internal: CFD8

reference

Port F, Chen HM, Lee T, Bullock SL.: Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):E2967-76.

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Tissue specific activity (Gal4/UAS)



name UAS-cas9.P/P2 description To express Cas9 under the control of the Gal4/UAS system we made two UAS-cas9 constructs. The first (called UAS-cas9.P on Flybase) contains a cas9 ORF that is codon optimised for expression in Drosophila and was cloned into the high-expression vector pJFRC81 (Addgene 36432). The second (UAS-cas9.P2 on Flybase) contains a human codon optimised cas9 (Addgene 41815) in a UAS vector containing a synthetic core promoter (Addgene 35200). Both constructs were inserted on attP40 on the second chromosome and attP2 on the third chromosome. genotype P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC]=UAS-Cas9.P}attP40

P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC]=UAS-Cas9.P}attP2

P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC]=UAS-Cas9.P2}attP40

P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC]=UAS-Cas9.P2}attP2

activity

All four UAS-cas9 stocks are highly active, with both UAS-cas9.P insertions showing higher activity than UAS-cas9.P2. We have found that the very high cas9 expression levels that result from expressing UAS-cas9.P in combination with strong Gal4 drivers causes substantial toxicity in flies. Furthermore, too high expression leads to ‘leaky’ mutagenesis outside the Gal4 expression domain. We therefore recommend to use the lower expressing UAS-cas9.P2 lines. These are good tools to induce mutations in a tissue specific manner.

stock ID

UAS-cas9.P at attP40: Internal: CFD4; Bloomington: 54594; VDRC:

UAS-cas9.P at attP2: Internal: CFD3; Bloomington: 54592; VDRC:

UAS-cas9.P2 at attP40: Internal: CFD9; Bloomington: 58985

UAS-cas9.P2 at attP2: Internel: CFD10; Bloomington: 58986

reference

The UAS-cas9.P lines have been described in:

Port F, Chen HM, Lee T, Bullock SL.: Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):E2967-76. The UAS-cas9.P2 lines are unpublished. Please acknowledge Fillip Port and Simon Bullock when using these lines.

name UAS-cas9.C description For this line a Drosophila codon-optimised cas9 with N- and C-terminal nuclear localisation signal was cloned into pJFRC28 (Addgene 36431) described in Pfeiffer et al., PNAS, 2012 (paper), using the KpnI and XbaI restriction sites. The Cas9 cDNA was a gift from Dr Justin Crocker (David Stern Lab, Janelia Farm, HHMI). The transgene is inserted at attP2 on the 3rd chromosome. genotype w1118; +; P(UAS-cas9, w+) attP2

activity

Kate has confirmed that this transgenic line is active.

stock ID

Internal: CFD5; Bloomington: 54595; VDRC: 300005

reference

This line is unpublished. If you publish work derived from the use of this fly line, please acknowledge Hui-Min Chen and Tzumin Lee (Janelia Farm, HHMI).

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High precision Cas9 variants



name act-Fok1:dCas9 description The Fok1 endonuclease domain was fused to a catalytically inactive Cas9 variant (D10A, H840A). The design of this construct is analogous to the one described by Keith Joung’s lab for expression in human cells (paper). The vector backbone is the same as used for CFD1 and contains an act5C promoter for strong ubiquitous expression. The plasmid is integrated at attP40 on the second chromosome. genotype P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC]=act-Fok1:dCas9}attP40

activity

We have tested this stock with two gRNA pairs and successfully mutagenized the yellow and ebony genes.

stock ID

Internal: CFD11; Bloomington: 58984

reference

The line is unpublished. Please acknowledge Fillip Port and Simon Bullock for sharing unpublished reagents.

name nos-Fok1:dCas9 description The Fok1 endonuclease domain was fused to a catalytically inactive Cas9 variant (D10A, H840A). The design of this construct is analogous to the one described by Keith Joung’s lab for expression in human cells (paper). The vector backbone is the same as used for CFD2 and contains an nos promoter and 3’UTR for germ line restricted expression. The plasmid is integrated at attP40 on the second chromosome. genotype P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC]=nos-Fok1:dCas9}attP40

activity

We have tested this stock with two gRNA pairs and successfully mutagenized yellow with one of them.

stock ID

Internal: CFD12; Bloomington: 58983

reference

The line is unpublished. Please acknowledge Fillip Port and Simon Bullock for sharing unpublished reagents.

name act-Cas9(D10A) (nickase) description This construct is similar to CFD1, except that one of the active sites was inactivated by a D10A mutation, thereby turning Cas9 into a nickase. It is integrated at attP2 on the 3rd chromosome. genotype P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC]=act-Cas9D10A}attP2

activity

We have confirmed the activity of this stock with one gRNA pair and successfully mutagenized yellow.

stock ID

Internal: CFD13

reference

Port F, Chen HM, Lee T, Bullock SL.: Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):E2967-76.

name vasa-Cas9(D10A) (nickase) description The line uses the Cas9 nickase from Feng Zhang’s lab (Broad Institute, Addgene plasmid 42333), which is tagged with HA. The coding sequence was cloned into a plasmid containing vasa regulatory elements (a gift from Johannes Bischof and Konrad Basler, paper) to confer germline specific expression and a 3xP3-EGFP marker. The plasmid is integrated at attP18 (Perrimon lab) on the X-Chromosome. genotype y1 w1118 P(3xP3-EGFP, vasa-cas9D10A)attP18

activity

Kate has confirmed that this transgenic line is active.

stock ID

Internal: CFD7

reference