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flyCRISPR header icon flyCRISPR Plasmids Available from Addgene


Harrison Lab - Wildonger Lab - O'Connor-Giles Lab email: [email protected]

We have found that a variety of Cas9-mediated genome modifications can be efficiently generated in Drosophila and transmitted through the germline. Using an injection approach, stable lines with targeted genome alterations can be generated within a month. phsp70-Cas9 and a single chiRNA plasmid can be coinjected to generate mutations via imperfectly repaired DSBs. Coinjection of phsp70-Cas9 and plasmids encoding two chiRNAs can induce defined deletions between the two cleavage sites, while the addition of an ssODN donor template can mediate gene replacement by homologous recombination.

For the sgRNA, vector pMB60 allows in vitro production of RNA from the T7 promoter, while pMB70 is designed for in vivo transcrition controlled by the regulatory sequences of an RNA polymerase III transcribed U6 snRNA. In both vectors, the target site sequence can be added by inserting an oligonucleotide linker into BsaI digested vector.

Cloning targeting chiRNAs

flyCRISPR chiRNA design cartoon

Targeting chiRNAs are easily cloned by annealed oligos into the pU6-BbsI-chiRNA plasmid via the BbsI restriction sites.

Design oligos based on the following template:

sense-antisense flyCRISPR cartoon

example :

lyCRISPR sequence cartoon

flyCRISPR bullet list

Resources:

These plasmids are described in:

Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Scott J. Gratz, Alexander M. Cummings, Jennifer N. Nguyen, Danielle C. Hamm, Laura K. Donohue, Melissa M. Harrison, Jill Wildonger and Kate M. O’Connor-Giles. Genetics. 2013 May 24. PubMed PMID 23709638. GENETICS.

Individual plasmids can be ordered via the links below:

ID Plasmid
46294 pBS-Hsp70-Cas9: A codon-optimized Cas9 nuclease under the control of the Drosophila hsp70 promoter.
45945 pHsp70-Cas9: The codon-optimized Cas9 nuclease under the control of the Drosophila hsp70 promoter used in Gratz, et al. (2013). Plasmid is low copy number.
45946 pU6-BbsI-chiRNA: Plasmid for expression of chiRNA under the control of the Drosophila snRNA:U6:96Ab promoter.
51019 pDsRed-attP: Vector for generating dsDNA donors for homology-directed repair to replace genes or other genomic sequence with an attP docking site. Contains the visible marker 3xP3-DsRed. (Also known as pHD-DsRed-att).
51026 U6-BbsI-crRNA: Generates crRNA for use in combination with tracrRNA.
51434 pHD-DsRed: vector for generating dsDNA donors for homology-directed repair. Contains the visible marker 3xP3-DsRed.