Supplementary Materials: <i>MFSD12</i> depletion reduces cystine accumulation without improvement in proximal tubular function in experimental models for cystinosis.
Creators: name: Bondue, Tjessa
name: Khodaparast, Laleh
name: Khodaparast, Ladan
name: Cairoli, Sara
name: Goffredo, Bianca Maria
name: Gijsbers, Rik
name: van den Heuvel, Lambertus
name: Levtchenko, Elena
Publisher: name: figshare
Year: 2024
Subjects: classificationCode: 320214
subject: Nephrology and urology

description: Supplementary MethodsSupplementary Method 1: Generation of CRISPRed MFSD12Cr/Cr ciPTECs by gene editingTwo MFSD12Cr/Cr ciPTEC cell lines were generated from the patient-derived ciPTECs using either a stably integrating lentiviral vector (LV) encoding SpCas9 and sgRNA (CYS ciPTEC MFSD12Cr/Cr – LV), or a virus-like particle (VLP) with the SpCas9 protein (CYS ciPTEC MFSD12Cr/Cr – VLP) and the guide RNA (5’-AAAGGCCAGGATCACCAGG-3’)(1). Briefly, LV genome contains a hU6 promoter-driven MFSD12 specific sgRNA (5’-AAAGGCCAGGATCACCAGG-3’) and a human elongation factor-core (EF1α-core) promoter driven SpCas9 coding sequence (CDS) together with a puromycin resistance cassette for selection purposes. LVs were produced by the Leuven Viral Vector Core as reported earlier(2). In parallel, we produced virus-like particles (VLPs) containing the SpCas9 protein and the sgRNA (5’-AAAGGCCAGGATCACCAGG-3’), that were produced as indicated in Mangeot et al.(3). The respective polyclonal MFSD12 CRISPRed cell lines were generated by serial diluting the LV on seeded ciPTEC cells in 96-well format. 72 hours after transduction, the cells were transferred to a 24-well format. Transduced cells were next selected using DMEM F-12 (Biowest, cat.no.L0093-500) supplemented with 1 µg/mL Puromycin (CAS 53-79-2, ThermoFisher Scientific). Surviving cells after selection with the lowest LV dose were expanded, which are presumed to have a single integrated copy for the vector. Likewise, a VLP dilution series in DMEM-F12 (Biowest, cat.no.L0093-500) was added to seeded ciPTEC cells in 96-well format and further expanded.Supplementary Method 2: gDNA analysis of CRISPR/Cas9 edit in the MFSD12 locusDry cell pellets were incubated for 3h at 55°C in lysis buffer (0.1M Tris HCl pH 8.5, 5mM EDTA pH 8.0, 0.2% SDS, 0.05M NaCl in water) with 0.1mg/ml Proteinase K. Exon 7 fragments were amplified for Sanger sequencing by thermal cycling (35x 30sec at 95°C, 55°C and 72°C) with 0.2µM MFSD12 gDNA specific primers (forward: 5’-AATAGCCACACGTCTCCAGCCGCT-3’, and reverse: 5’-TTCTGTGGAGACACAGGCGA-3’) and 1X GoTaq G2 Master Mixes (Promega, cat.no.M7823). Next, DNA was purified with PureIT ExoZAP PCR CleanUp (Ampliqon, cat.no. 75001) and purified products were prepared for sequencing through the Big Dye Terminator technology. Sequencing was performed by the Centre of Human Genetics at the University Hospitals Leuven. Deconvolution and frameshift score prediction was performed with the DECODR(4) algorithm for a range of 10 passages after generation of the cell models.Supplementary Method 3: RNA isolation, cDNA synthesis and (quantitative) PCRThe ciPTECs were transferred to a 37°C incubator to allow differentiation. After 10 days, the cells were collected by trypsinization (Life Technologies, cat.no. 25200056). RNA was purified from the pellets with the Qiagen RNeasy Plus Micro Kit (Qiagen, cat.no. 74034) according to the manufacturers protocol. The concentration was determined with the ND1000 spectrophotometer from Isogen Life Science (The Netherlands). cDNA was prepared with a mix of Oligo (dT) (Thermofisher, cat.no.18418-012), random primers (Thermofisher, cat.no.48190-011), dNTP mix (VWR, cat.no.733-1364) and SuperScript-III Reverse Transcriptase (Invitrogen, cat.no. 18080-085), according to the manufacturer’s protocol. The stable cDNA was used for q-RT-PCR as described previously(5). MFSD12 primers were used for the quantification of the MFSD12 knockout (forward: 5’-TCCACACAGATCTCCCACCT-3’, and reverse: 5’-GCCGTAGACGGTGATGTTG-3’) and GAPDH was used as a housekeeping gene (forward: 5’-TGGAAGGACTCATGACCACAG-3’, and reverse: 5’-CATCACGCCACAGTTTCCC-3’).PCR in zebrafish embryos was utilized to observe the splice modification after the injection of 200µM splice-blocking morpholino (SB MO). The zebrafish were collected (9 embryos per replicate) at 24h and 72h post-injection of the SB MO and RNA isolated. Collected embryos were sonicated in 1 ml of Trizol (Thermofisher, cat.no.15596026) and phase separation was performed with chloroform (Sigma, C2432), according to the manufacturer’s protocol. cDNA was prepared as described above. A PCR mixture of 50 ng gDNA and 50% GoTaqGreen (Promega, USA, cat.no. M7823) in water was prepared. 200µM of primers are added (Forward: 5’-ACGGTCAGGTCATCACCAT-3’ and Reverse: 5’-AGGGTACATGGTGGTACCTC-3’ for bactin1, Forward: 5’-ACAGTGGGTCAGTGTCACCTAT-3’and Reverse: 5’-GAGCCAGTTATTCCAGTGCAGTAG-3’ for mfsd12a). A PCR with 35 cycles of 30 sec at 94°C, 54 °C, and 72 °C, was performed and the products were subsequently separated on an 2% Agarose gel (90V, 35min) for imaging with the Syngene Chemi XRQ. Band intensity was quantified with ImageJ software.Supplementary Method 4: Cystine measurementCystine levels were measured, as described previously(5–8), in a total volume of 200µL 50mM N-ethylmaleimide (Sigma, cat.no.04259) and 100µL of 12% sulfosalicylic acid (Sigma, cat.no.S7422). For cystine measurement in zebrafish larvae, embryos were collected in groups of 10-12 fish at 72h post-injection and washed with methylene blue-free aquarium water first(9). Cystine was measured by liquid chromatography coupled to tandem mass spectrometry (‘LC-MS/MS’) using the UHPLC Agilent 1290 Infinity II 6470 (Agilent Technologies) equipped with an ESI-JET-STREAM source operating in the positive ion (ESI+) mode. Concentrations were expressed as nmol cystine/mg protein after normalization to total protein, which was measured by means of a Bicinchoninic acid assay (Thermofisher, cat.no. 23225)(7, 8).Supplementary Method 5: Cryosection and immunostaining of zebrafish larvaeFish were fixed overnight in 4% PFA and prepared for cryosection as described previously(5). Sections were made with the NX70 cryostat (4µm, Thermofisher, USA) and blocked by overnight incubation in PBS + 0.1% Triton X-100 and 5% donkey (Abcam, cat.no.ab7475), followed by overnight incubation with primary antibody (Homemade in University of Manchester, Prof. Martin Lowe, 1:200) and 2h with secondary Alexa-488 labelled donkey anti-rabbit (Abcam, cat.no.ab150073, 1:500). Sections were stained with Hoechst33342 and mounted with fluorescence mounting medium (DAKO, s3023). Megalin was imaged with the Leica DMI6000 B inverted fluorescence microscope (Leica Microsystems, Germany – Laboratory of Angiogenesis and Vascular Metabolism (VIB-KU Leuven)).Supplementary Figure 1: mfsd12a splice blocking (SB) morpholino in ctns-/- embryos only results in partial mfsd12a knockdown and does not reduce cystine levels. ctns-/- embryos were injected at the one-cell stage with 200µM of mfsd12a splice-blocking morpholino (SB MO). (A/B) RNA was isolated and cDNA prepared from a group of 9 embryos at 24h and 72h post-injection of 200µM SB MO (lane 2 and 3) or from untreated ctns-/- zebrafish (lane 1). A PCR with bactin1 (A) and mfsd12a (B) specific primers was executed and the products were separated on an 2% Agarose gel. The native (normally spliced) mfsd12a mRNA results in a 451bp band, which can be observed in the untreated ctns-/- (lane number 1) embryos and those treated with the SB MO (lane number 2 and 3, each representing a separate group of 9 SB MO treated fish). In the SB MO treated ctns-/-embryos, the second band represents the spice-modified amplicon (&gt;500bp). NC = no DNA. (C) To quantify the proportion of wildtype and alternatively spliced products formed, the band intensity for each condition was analysed (normalized to the bactin1 signal). As expected, lane 1 (untreated fish) showed that only the normal splice product is formed (approximately 100% of signal). Lane 2 and 3 represent a biological duplicate and show that the &gt;500bp fragment is formed, but that there is still a remaining signal of the 451bp product. Notably, the approximately 50% of 451bp product at 72h suggests that the proportion of native mfsd12a increased over time after treatment with the SB MO. Medians with 95% CIs are shown (n=2).(D) In parallel, cystine levels were determined in the mfsd12a SB MO treated embryos (15 fish per group, representing one biological replicate) in comparison to the wildtype (ctns+/+) control, the untreated ctns-/- embryos and the fish treated with the translation blocking morpholino (+200µM TB MO). This demonstrated only a small decrease in cystine levels after treatment with the SB MO, in comparison with the TB MO that was more efficient at reducing cystine levels (n=1 for each ctns-/- group, n=3 for ctns+/+).<br><br><br>