Cloning and plasmids

A split mVenus system was a gift from the Skehel lab [22]. From this, we generated a single ER-Mito reporter construct: Each of the two fragments (TOMM20-mVenus and VAPB -mVenus) of the split mVenus system were amplified via PCR using specific Gibson assembly primers that also incorporated a self-cleaving T2A site between the two fragments. The backbone is based on a doxycycline NGN2 inducible construct [77]. The backbone consisted of a 3^rd generation TET-ON system, allowing temporal control via doxycycline, a LoxP flanked removable mCherry-Puromycin for clonal selection and AAVS1 safe harbour homology arms for incorporation into the AAVS1 safe harbour locus. NGN2 was removed via restriction enzyme digest, and the split mVenus constructs with homology arms were combined using Gibson assembly mix (NEB, E2611) for 1 h(h) at 50 °C. These were transformed into NEB 10-beta competent E. coli, colonies picked, and Sanger sequenced to confirm insertion of the full-length reporter construct.

The top 3 gRNAs of each gene were selected from the pooled screen, and the sequences were acquired. For each gRNA, a forwards and reverse oligo was designed, including specific BBS1 complementary overhang sequences. Forwards (caccg- gRNA-gt) Reverse (ttaaac-gRNA-c). The forwards and reverse primers were annealed and then ligated into the BBS1 cut U6-sgRNAv2-ccdb-BFP-PURO plasmid. These were then transformed into NEB 10-beta competent E. coli (NEB, C3019I) using 20 µL of competent cells per well in a 96-well clear round bottom deep well plate (Axygen, P-2ML-SQ-C) by applying heat shock (1 min at 42 °C) followed by incubation for 1 h at 37 °C with 1 mL of SOC media. Twenty microlitres of the transformation mix was transferred to 24 deep-well culture plates (Axygen, 12537837) with 5 mL media and incubated overnight for 24 h at 30 °C. Cells were pelleted, and DNA was extracted in 96-well plates. DNA was quantified using Quant-iT™ PicoGreen™ (Thermo, P7589) and normalised to 10 ng/µL ready for use in virus generation.

HeLa cells were maintained at 37 °C with 5% CO₂ in a tissue culture incubator in minimum essential medium (MEM) + Glutamax (Gibco, 42360-032) substituted with 10% fetal bovine serum (FBS) (Sigma, F9665) and 1× nonessential amino acids (NEAA). A total of 1.8 µg of Cas9-Blast PiggyBac and 200 ng of transposase (pBac) were electroporated into the HeLa cell line using the Amaxa SF Cell line Nucleofector kit (Lonza, V4XC-2012). Cas9-positive cells were selected using 10 µg/mL blasticidin S HCl (blast) (Gibco, A11139-03) over 10 days to generate a pool of HeLa Cas9-blast cell lines. A gRNA targeting the AAVS1 safe harbour (GGG GCC ACU AGG GAC AGG AU), 2 µg donor ER-Mito mCherry Puromycin reporter construct and 20 µg of Alt-R™ S.p. HiFi Cas9 Nuclease (IDT, 1081060) was nucleofected using the Amaxa SF Cell line Nucleofector Kit (Lonza, VCA-1003) and placed at 32 °C for 48 h. ER-Mito mCherry-positive cells were selected using 5 µg/µL puro. Cells were maintained in Puromycin (Gibco, A1113803). and blast to generate a Cas9-blast mCherry-puro population. The ER-Mito mCherry-Puro pool was then treated with Cre recombinase to remove the Lox P-flanked mCherry-puro. Clones were picked, and their DNA was extracted before storage. The clones were genotyped using two primer pairs (Supplementary Fig. 1a) that can detect a loss of the Lox-P-flanked mCherry-puro region. The PCR products of 3 clones along with genomic DNA from control cells (con) that were not treated with Cre and still contained the mCherry-Pruo construct were compared. With primer set 1, there was a shift in the band, while primer set 2 showed a loss of a PCR product, showing that mCherry-puro was removed (Supplementary Fig. 1a, b). Flow cytometry was used to select mVenus-positive and mCherry-negative cells for expansion and further testing (Supplementary Fig. 1c). Clones 1 and 2 were taken forwards, but Clone 3 was not. To determine the levels of mVenus fluorescence that would be suitable for our screen, we performed a time course analysis of doxycycline induction (Supplementary Fig. 2a–d). We found that treating ER-Mito reporter cells with doxycycline for 24 h, followed by its removal for an additional 48 h, resulted in levels of mVenus fluorescence that were lower than the maximum observed levels at 24 h, indicating that the fluorescence at 48 h was not saturated.

Flasks (pooled gRNA library) and plates (arrayed gRNA library) were coated with 25 µg/mL PDL (Gibco, A38904-01) for 3 h or overnight, washed with PBS and Hek cells plated at 80–90% confluence. Transfection was conducted using Lipofectamine LTX (Invitrogen, 15338) reagent as per these ratios for one well of a 96-well plate. Reaction A (20 µl Optimem, 19.12 ng psPAX2, 12.5 ng pMD2. G, 0.1 µL PLUS reagent) and 25 ng gRNA plasmid combined with Reaction B (5 µL optimem and 0.3 µL Lipofectamine LTX). Plates or flasks were spun down (300 × g for 5 min), and the medium was aliquoted (arrayed gRNA Library) or centrifuged at 7000 g at 4 °C overnight, and the pellet was resuspended in PBS.

Pooled next-generation sequencing (NGS) screening was conducted as described in the results and depicted in Fig. ​Fig.2a.2a. Once FAC sorted via their MedFI, the DNA was extracted from each population. Cells were lysed for 4 h at 55 °C in DNA lysis buffer (50 mM Tris pH 8, 100 mM NaCl, 10 mM EDTA, 1% SDS and 0.5 mg/ml proteinase K (Thermo, EO0491)). After 4 h, DNA was precipitated using an equal volume of isopropanol and resuspended in TE. The gDNA was quantified using quBit, and the whole sample was used in multiple PCRs to enrich gRNA cassettes (2 µg DNA, 1.5 µL 10 µM primers, 25 µL Q5 master mix (NEB, M0492S)).

The PCRs were pooled, bead purified, and the product was used in a second PCR to add index adaptors (25 µL KAPA HiFi Hot start ready mix, 1 µL 10 µM primers i5 and i7 combinations). The product was quantified using the NEBNext Library Quant Kit for Illumina (NEB, E7630S) diluted to 4 nM and sequenced by NextSeq (Illumina) using the manufacturer’s instructions for NextSeq 500/550 High Output Kit 75 Cycles (Illumina, 20024906).