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
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:
- Protocol for generating gRNAs 106.5 KB
- flyCRISPR website contains additional information
- CRISPR Optimal Target Finder Identifies gRNA target sequences from an input sequence and checks for off-target binding. Currently supports over 20 model and non-model invertebrate species.
These plasmids are described in:
Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O'Connor-Giles KM. Genetics. 2013 May 24. doi: 10.1534/genetics.113.152710 (Link opens in a new window) Pubmed (Link opens in a new window) Article
Individual plasmids can be ordered via the links below:
|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.|