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CRISPR: Drosophila


The following CRISPR plasmids have been designed for use in Drosophila and other insects.

Cut

Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism which frequently causes insertions or deletions (indels) in the DNA. Indels often lead to frameshifts, creating loss of function alleles.

To introduce specific genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and a specific edit close to the gRNA PAM site. When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead of NHEJ. In most experimental systems, HDR occurs at a much lower efficiency than NHEJ.

Plasmid Gene/Insert Promoter PI Publication Hidden Extra Search Info
pHsp70-Cas9Codon optimized Cas9 O'Connor-Giles Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics. 2013 May 24. The codon-optimized Cas9 nuclease under the control of the Drosophila hsp70 promoter used in Gratz, et al. (2013). Plasmid is low copy number. pHSS6
pBS-Hsp70-Cas9codon optimized Cas9Hsp70 O'Connor-Giles FlyCRISPR (unpublished) A codon-optimized Cas9 nuclease under the control of the Drosophila hsp70 promoter. pBluescript-KS(+)
pAc-sgRNA-Cas9Cas9 (Synthetic), dU6-sgRNA (Drosophila melanogaster)Actin-5c, Drosophila U6 Liu Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9. Biol Open. 2013 Dec 10. pii: bio.20137120v1. doi: 10.1242/bio.20137120. Expresses sgRNA and Cas9-Puro in Drosophila S2 cells pAc-STABLE1-Puro
pRB14S. pyogenes cas9 with humanized codon bias (Other)tubulin Foerstemann Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells. Nucleic Acids Res. 2014 Apr 19. expresses a myc-tagged version of hCas9 in Drosophila pCASPER5
pDCC6Cas9 (Synthetic), U6-2>sgRNA (Synthetic)hsp70Bb, U6-96Ab Duchek Efficient CRISPR/Cas9 Plasmids for Rapid and Versatile Genome Editing in Drosophila. G3 (Bethesda). 2014 Sep 17. pii: g3.114.014126. doi: 10.1534/g3.114.014126. Bi-cistronic Drosophila CRISPR/Cas9 vector, contains hsp70>Cas9 and U6>sgRNA cassette pHW
pnos-Cas9-noscas9nanos Bullock Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A. 2014 Jul 7. pii: 201405500. Expresses Cas9 under control of nanos promoter and 3'UTR. For germ line restricted genome engineering in Drosophila melanogaster. pnos
pAct-Cas9cas9act5C Bullock Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A. 2014 Jul 7. pii: 201405500. Expresses Cas9 under control of act5C promoter and SV40 3'UTR. For ubiquitous genome engineering in Drosophila melanogaster. pact
p(bhsp68-Cas9)Cas9 (Other)bhsp Averof Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum. Development. 2015 Jul 9. pii: dev.125054. Tribolium basal heat shock promoter driving Cas9 pSLfa[Tc-bhsp68::nlsEGFP]fa
act-AsCpf1hAsCpf1 (Homo sapiens)act5C Bullock Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nat Methods. 2016 Oct;13(10):852-4. doi: 10.1038/nmeth.3972. Epub 2016 Sep 5. expression plasmid of AsCpf1 pact

Nick

CRISPR/Cas nickase mutants introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type Cas enzymes. To use a nickase mutant, you will need two gRNAs that target opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB) that is repaired using error-prone non-homologous end joining (NHEJ). Double nicking strategies reduce unwanted off-target effects. Nickase mutants can also be used with a repair template to introduce specific edits via homology-directed repair (HDR).

Plasmid Gene/Insert Promoter PI Publication Hidden Extra Search Info
pDCC5Cas9-D10A (Synthetic)hsp70Bb Duchek Efficient CRISPR/Cas9 Plasmids for Rapid and Versatile Genome Editing in Drosophila. G3 (Bethesda). 2014 Sep 17. pii: g3.114.014126. doi: 10.1534/g3.114.014126. Bi-cistronic Drosophila CRISPR/Cas9 vector, contains hsp70>Cas9-D10A nickase and U6>sgRNA cassette pHW

Activate

Catalytically dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator to promoter or regulatory regions of your gene of interest. If the plasmid that you choose does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9-activator to your specific locus.

Plasmid Gene/Insert Promoter PI Publication Hidden Extra Search Info
pAWG-dCas9-VPRSP-dCas9-VPR (Drosophila melanogaster)act5c Church Highly efficient Cas9-mediated transcriptional programming. Nat Methods. 2015 Mar 2. doi: 10.1038/nmeth.3312. SP-dCas9 with VP64-p65-Rta (VPR) fused to it's C-terminus; drosophila vector pAWG
pWalium20-10XUAS-3XFLAG-dCas9-VPRdCas9-VPR (Homo sapiens)10XUAS Perrimon In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila. Genetics. 2015 Oct;201(2):433-42. doi: 10.1534/genetics.115.181065. Epub 2015 Aug 5. Expresses 3XFLAG-dCas9-VPR (human codon) under 10XUAS control, for in vivo CRISPRa pWalium20
pAct:dCas9-VPRdCas9-VPRpActin (Drosophila) Perrimon Comparison of Cas9 activators in multiple species. Nat Methods. 2016 May 23. doi: 10.1038/nmeth.3871. Expresses dCas9-VPR under Actin promoter for CRISPRa in Drosophila cell culture. Please note- this plasmid does not express GFP. pAWG
pAct:dCas9-GCN4dCas9-10XGCN4pActin (Drosophila) Perrimon Comparison of Cas9 activators in multiple species. Nat Methods. 2016 May 23. doi: 10.1038/nmeth.3871. Expresses dCas9-GCN4 under actin promoter for SunTag CRISPRa in Drosophila cell culture pAWG
pAct:dCas9-VP64dCas9-VP64pActin (Drosophila) Perrimon Comparison of Cas9 activators in multiple species. Nat Methods. 2016 May 23. doi: 10.1038/nmeth.3871. Expresses dCas9-VP64 under actin promoter for SAM CRISPRa in Drosophila cells pAWG

Empty gRNA Expression Vectors

Select a gRNA expression plasmid based on factors such as selectable marker or cloning method. When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function is often limited to a single category (cut, nick, etc.) On the other hand, gRNA plasmids that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids.

gRNA Plasmid Promoter Cloning
Enzyme(s)
Delivery Resistance Co-expressed Cas9 Depositing lab
Cas9 species = S. pyogenes (PAM = NGG)
pCFD1-dU6:1gRNA dU6:1 BbsI Injection or in vitro transcription Virmilion none, need Cas9 plasmid Bullock and Port
pCFD2-dU6:2gRNA dU6:2 BbsI Injection or in vitro transcription Virmilion none, need Cas9 plasmid Bullock and Port
pCFD3-dU6:3gRNA dU6:3 BbsI Injection or in vitro transcription Virmilion none, need Cas9 plasmid Bullock and Port
pCFD4-U6:1_U6:3tandemgRNAs dU6:1 and dU6:3 BbsI Injection or in vitro transcription Virmilion none, need Cas9 plasmid Bullock and Port
pAc-sgRNA-Cas9 dU6 BspQI Transfection Puromycin yes, cut Ji-Long Liu
U6-BbsI-crRNA dU6 BbsI Transfection none, need Cas9 plasmid O'Connor-Giles,
Harrison, Wildonger
pU6-BbsI-chiRNA dU6 BbsI Transfection none, need Cas9 plasmid O'Connor-Giles,
Harrison, Wildonger

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