The CRISPR system allows investigators to target double-stranded DNA breaks to specific genomic sequences. A trans-activating RNA (tracrRNA) interacts with a Cas nuclease (eg., Cas9, Cas12a) to direct cleavage to a sequence complementary to an introduced RNA (CRISPR RNA or guide RNA).
Detailed information about the CRISPR system in Drosophila is available from the flyCRISPR, FlyCas9 and CRISPR fly design websites. For a nice review of CRISPR technology with helpful definitions and schematics, see Zirin et al., 2022.
We also include stocks expressing dCas9.VPR on this page. dCas9.VPR is a nuclease-dead Cas9 fused to a chimeric transcription activator. When targeted to the 5' end of a gene with a gRNA, it can activate transcription of the gene.
See also pages for: P{white-eraser} - for replacing a mini-w+ marker with 3xP3-RFP DNA base editors - for creating point mutations in DNA
This stock ubiquitously expresses a gRNA that targets Cas9 for Cas9-triggered chain ablation (CATCHA). In the presence of a Cas9 transgene, the CATCHA gRNA cleaves Cas9 sequences and subsequent homology directed repair should result in conversion of Cas9 to CATCHA, rendering it inactive. For more information see Wu et al., 2016.
P{nos-FokI.Cas9} and P{Act5C-FokI.Cas9} express the FokI endonuclease domain fused to catalytically inactive Cas9 in the ovary in the pattern of the nos gene or ubiquitously in the pattern of the Act5C gene. FokI must dimerize to cleave DNA; consequently, the Fok1-dCas9 fusion protein leads to heritable genome edits only when recruited by two guide RNAs targeting sequences flanking the cleavage site in a defined spacing and orientation. The specificity of DNA cleavage by the fusion protein is better than that obtained with wild type Cas9.
These stocks express Cas9 protein fused to the ey DNA-binding domain under the control of Act5C regulatory sequences. The ey DBD helps recruit 3xP3-containing DNA fragments to Cas9-induced breaks and enhance their incorporation into the site upon DNA repair.
These stocks carry Acidaminococcus Cas12a RNA-guided endonuclease (aka Cpf1) under the control of Act5C regulatory sequences. Cas12a enzymes require a different PAM sequence than Cas9, make staggered DNA cuts and have an RNase activity that can cut single crRNAs from crRNA arrays.
All stocks carrying transgenes expressing the Cas9 (active) or dCas9 (inactive) are listed here.
Bloomington Drosophila Stock Center
Dept Biology, Indiana University 1001 E. Third St. Bloomington, IN 47405-7005 USA
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