Polarized transport is essential for the construction of multiple plasma membrane domains within cells. Drosophilaphotoreceptors serve as excellent model systems for studying the mechanisms of polarized transport. We conducted a comprehensive SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) screening of the fly genome using RNAi knockdown and CRISPR/Cas9 somatic knockout combined with the CoinFLP system to identify SNAREs involved in post-Golgi trafficking. The results suggest that, in post-Golgi transport, no SNARE is exclusively responsible for transport to a single specific plasma membrane domain. However, each SNARE shows some preference for certain membrane domains: the loss of nSyb, Ykt6, and Snap24/25 results in severe defects in rhabdomere transport, while the loss of Syx1A and Snap29 leads to significant impairments in basolateral transport. Together with the function of Syx1A, Snap25, and nSyb in the fusion of synaptic vesicles with the synaptic plasma membrane, these results suggest that SNAREs are not the sole determinants for vesicles to specify their target subdomains in the plasma membrane. Furthermore, rhodopsin transport to the rhabdomere requires two kinds of R-SNAREs, Ykt6 and nSyb, suggesting that multiple sets of post-Golgi SNAREs are contributing in tandem or in cooperation, rather than in parallel.

Plasmids were constructed as follows. 

pU62-wsh1
pU62-wsh1 is a plasmid carrying a cassette that works as a "dominant white-eye" marker in Drosophila, consist of Drosophila U6-2 promoter and shRNA against white gene. This vector was constructed based on a vector, pAc-sgRNA-Cas9 (Bassett et al., 2014) by inserting a synthetic shRNA unit against white gene, HMS00017, predesigned in TRiP RNAi & CRISPR fly project (Zirin et al., 2020). Four oligonucleotides TTC-wsh1-F1, wsh1-R1p, wsh1-F2p and wsh1-Ap-GTT were ligated into pAc-sgRNA-Cas9 digested with BbsI, to obtain pU62-wsh1.

pBIDwsh-UASC
pBIDwsh-UASC is a phi31 transgenic vector based on pBID-UASC (Wang et al., 2012) but carrying "dominant white-eye" marker instead of miniature-white “red-eye” marker. From pU62-wsh1, U6-wshRNA cassette was amplified with primers Nh-U62 and gCas9puro-F, then digested with NheI and ApaI, and then cloned between NheI and ApaI sites of pBID-UASC to obtain pBID-wsh-UASC.

pBIDwsh-Act-coinFLP-Gal4
pBIDwsh-Act-coinFLP-Gal4 is a phi31 transgenic vector carrying "dominant white-eye" marker and coinFLP-Gal4 cassette under control of Act5C promoter. From original coinFLP vector, pAct-FRT-stop-FRT3-FRT-FRT3-Gal4 attB (Bosch et al., 2015), the DNA fragment including Act5C promoter and FRT-stop-FRT3-FRT-FRT3-Gal4 cassette was amplified with primers Sbf-BID-Act-F and Gal4-BID-Xh-R. Using Gibson assembly, the fragment was cloned between SbfI and BglII sites of pBIDwsh-UASC, replacing UASC promoter and multiple cloning sites, to obtain pBIDwsh-Act-coinFLP-Gal4.

5x longGMR promoter
The longGMR promoter (Wernet et al., 2003) consists of five copies of enhancer sequence in the upstream of Rh1 promoter and a basal promoter core of hsp70. Since the detail of the construct was not described, the region containing longGMR promoter was amplified using PCR with primers wdelR_Nhe and Gal4DBD-R2 from a transgenic fly caring P{longGMR-Gal4} (Bloomington Drosophila Stock Center #8605)..
pP-longGMR, pBID-longGMR and pBIDwsh-longGMR
The fragment containing longGMR promoter was digested with SphI and EcoRI and then ligated between SphI and EcoRI sites of pPdM-UAST (Yamashita et al., 2022), pBID-UASC, and pBIDwsh-UASC, to obtain pP-longGMR, pBID-longGMR, and pBIDwsh-longGMR, respectively.
pP-3xlongGMR, pBID-3xlongGMR and pBIDwsh-3xlongGMR
DNA fragments with various number of LGMR repeats were amplified from pP-longGMR, with primers Sph-longGMR and UAST-R4. The fragments were digested with SphI and EcoRI, then separated by size in agarose gel electrophoresis, and then ligated into pPdM-UAST, pBID-UASC, and pBIDwsh-UASC, to obtain pP-3xlongGMR, pBID-3xlongGMR, and pBIDwsh-3xlongGMR, respectively.
pP-longGMR-coinFLP-Gal4 and pBIDwsh-longGMR-coinFLP-Gal4
From pBIDwsh-Act-coinFLP-Gal4, DNA fragments including FRT-stop-FRT3-FRT-FRT3-Gal4 cassette were excised with BamHI and KpnI, and then ligated between BglII and KpnI sites in pP-longGMR, pP-3xlongGMR and pBIDwsh-longGMR to obtain pP-LongGMR-coinFLP-Gal4, pP-3xlongGMR-coinFLP-Gal4 and pBIDwsh-longGMR-coinFLP-Gal4.

pX-HFCas9
A mammalian CRISPR-Cas9 KO vector, pX-HFCas9, coding HiFiCas9R691A (Vakulskas et al., 2018) was constructed based on pSpCas9(BB)-2A-Puro PX459 V2.0 (Ran et al., 2013), by introducing R691A mutation in the Cas9 gene using Gibson assembly with primers HF-Cas9-F and HF-Cas9-R.
pBIDwsh-Act-coinFLP-HFCas9-H2B-2xNG and pP-3xlongGMR-coinFLP-HFCas9-H2B-2xNG
DNA fragments including HFCas9-T2A was amplified from pX-HF-Cas9 with Bg-Coin-Cas9 and ddT2A-R. DNA fragments including H2B-GFP was amplified from pcDNA3.1 miniSOG2 T2A H2B-EGFP (Makhijani et al., 2017) with ddT2A-F2 and H2B-EGFP-BID-K. Using Gibson assembly, fragments HFCas9-T2A and H2B-2xmNeonGreen were cloned between BglII and KpnI sites of pBIDwsh-Act-coin-Gal4 and pP-3xlongGMR-coinFLP-Gal4 to obtain pBIDwsh-Act-coinFLP-HFCas9-H2B-2xNG and pP-3xlongGMR-coinFLP-HFCas9-H2B-2xNG.

pCFD4w-Syx6
A phi31 vector pCFD4w was constructed by replacing promoter-gRNA scaffold of pCFD5w, a phi31 vector with white+ marker (gift from Michael Boutros, Addgene plasmid #112645), by those of pCFD4. DNA fragments including dU6:2-BbsI-gRNA scaffold were amplified with primers Sgf-U61F and dU6-wsh-fix and digested with EcoRI and NheI, then inserted between EcoRI and XbaI sites of pCFD5w. To construct pCFD4w-Syx6, oligonucleotides Syx6-gRNA3f and Syx6-gRNA3r were annealed and ligated with BbsI digested pCFD4w.

pUAST-myc-Ykt6, pUAST-HA-Snap24 and pUAST-HA-Snap25 
From third instar larvae cDNA, DNA fragment encoding Ykt6 were amplified using primers Xh-dYkt6 and dYkt6-Mlu, then digested with XhoI and MluI, and then ligated between XhoI and MluI sites in pMT-HA-NBgA-m, to generate pMT-HA-Ykt6. The fragment containing Ykt6 was excised with XhoI and XbaI and then inserted into pPdM-UAST-m2NBgAm to generate pUAST-myc-Ykt6. DNA fragment encoding snap24 and snap25 were amplified using primers Xh-Snap24, Snap24-Ap, Xh-Snap25 and Snap25-Ap, digested with XhoI and ApaI, and cloned into pMT-HA-NBgA-m to obtain pMT-HA-snap24 and pMT-HA-snap25. HA-snap24 and HA-snap25 fragments were excised with KpnI and MluI, and then inserted into pPdM-UAST-m2NBgAm to generate pUAST-HA-Snap24 and pUAST-HA-Snap25.