Fig. 2: Targeted library CRISPRi screen confirms SLC25A38 as a key B6 essential gene in K562 cells and highlights the importance of mitochondrial PLP-dependent metabolism.

A Gene Scores (GS) for the targeted library screen in -B6 or +B6 conditions. B Top differentially essential genes with reduced fitness in B6 stress, with SLC25A38 showing the highest delta Gene Score (dGS: GS in +B6 minus GS in -B6). C Guide scores for SLC25A38 in the genome-wide and targeted CRISPRi screens. D Diagram depicting the PLP-dependent glycine metabolism pathways in mitochondria and cytosol; PLP, pyridoxal 5′-phosphate; CH2-THF, Methylenetetrahydrofolate; 5-ALA, aminolevulinic acid; SHMT, serine hydroxymethyltransferase; GCV, glycine cleavage system (P-protein); ALAS2, Delta-aminolevulinate synthase 2; GCAT, Glycine C-acetyltransferase; PSAT1, phosphoserine aminotransferase 1; SFXN1, sideroflexin 1; ABCB6, ATP-Binding Cassette Sub-Family B Member 6; NFS1, cysteine desulfurase; ISC, iron-sulfur clusters. E Proliferation in B6 rich or B6 poor conditions for clonal knockout cell lines of SLC25A38, SLC25A39, PDXK, and PROSC. Proliferation defects were observed in PDXK, PROSC, and SLC25A38 knockouts in -B6, but not in WT and SLC25A39 knockouts. Cells were plated for proliferation assays after 3 days of conditioning in each respective media. Doublings are shown as mean ± SD; n = 3 independent cell cultures (E), two-way ANOVA followed by Šídák post hoc analysis (E) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Each n was defined as an independent culture of cells. F K562 cells were conditioned in B6-depleted media supplemented with low B6 (1 nM PN), physiological B6 (10 nM PN), or high B6 (1000 nM PN) for a longer period (6 days, media change every 2 days) and then plated onto 96 well plates at 10.000 cells per well (two replicates) for growth over 4 days in eight media conditions: B6-depleted media without supplementation, +0.5 nM PN, +1 nM PN, +5 nM PN, +10 nM PN, +50 nM PN, +100 nM PN and +1000 nM PN (“PN added to B6-depl.”). Doublings were estimated from absorbance reads using the Presto Blue assay at day 3 (E) or day 4 (F). D Created in BioRender. Pena, I. (2025) https://BioRender.com/m62v944. Source data are provided as a Source Data file.