CRISPR system stocks for site-specific mutagenesis

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 guideRNA).

Detailed information about the CRISPR system in Drosophila is available from the flyCRISPRFlyCas9 and CRISPR fly design websites.

Tools for designing guide RNAs: Find CRISPRs from the TRiP, Optimal Target FInder from flyCRISPR, Cas9 Target Finder from FlyCas9

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 RNAase activity that can cut single crRNAs from crRNA arrays.