Pact cas9

Plasmids were prepared using the Qiagen Plasmid Midi kit (#12191), and sequence was checked. Each gRNA construct was coinjected with a transient source of Cas9 (pAct-Cas9, Addgene plasmid no. 62209) by Rainbow Transgenics. Injection mixes were assembled with each gRNA plasmid (final concentration: 500 ng/μl) and pAct-Cas9 (final concentration: 500 ng/μl) in a volume of 50 μl. Injection mixes for all gRNA constructs were injected into an Oregon-R (white⁺) stock (Bloomington Drosophila Stock Center, BDSC #2376). White⁻ mutant males were selected in F1 progeny to establish isogenic lines, and specific alterations were determined by sequencing.

We used the following plasmids that were available from Addgene: pAct:Cas9 (62209) for generating spok_HsC, pAct:dCas9-VP64 (78901) for generating spok_64aO, spok_64bO and spok_dI, pWalium20-10xUAS-3xFlag-dCas9-VPR (78897) for generating spok_VPRO, pP(ELAV-Geneswitch) (83957) for generating spok_GSO, pBPGUw (17575) for generating gG.Cas9 collection, pUC19 (50005), pCFD3 (49410), pCFD4 (49411) and pCFD5 (73914) (Ornitz et al., 1991 (link); Wang et al., 2012 (link); Port et al., 2014 (link); Lin et al., 2015 (link); Chavez et al., 2016 (link); Port and Bullock 2016 (link)). pAct:FokI-dCas9 (62211) for generatings spok_dI was a kind gift from Simon Bullock’s lab. pVasa.Cas9 (1340) was obtained from Drosophila Genomics Resources Center for generating spok_DmC. All PCR reactions were performed using NEB Q5 high fidelity DNA Polymerase (NEB M0491S) and purified by HighPrep PCR reagent from MagBio (AC-60005) following manufacturer’s protocol. All cloning steps were based on the Gibson reaction (Gibson et al., 2009 (link)).