DOI: 10.5061/dryad.wm37pvms7
title: CRISPR/Cas9 screen for essential endolysosomal genes under free choline limitation

description: Lysosomes degrade macromolecules and recycle their nutrient content to
 support cell function and survival. Yet, the machineries involved in
 lysosomal recycling of many nutrients remain to be discovered, with a
 notable example being choline, an essential metabolite liberated via
 phospholipid degradation. Here, we engineered metabolic dependency on
 lysosome-derived choline in pancreatic cancer cells to perform an
 endolysosome-focused CRISPR-Cas9 screen for genes mediating lysosomal
 choline recycling. We identified the orphan lysosomal transmembrane
 protein SPNS1 as critical for cell survival under choline
 limitation. SPNS1 loss leads to intralysosomal
 accumulation of lysophosphatidylcholine (LPC) and
 lysophosphatidylethanolamine (LPE). Mechanistically, we reveal that
 SPNS1 is a proton gradient-dependent transporter of LPC species
 from the lysosome for their re-esterification into phosphatidylcholine in
 the cytosol. Finally, we establish that LPC efflux by SPNS1 is required
 for cell survival under choline limitation. Collectively, our work defines
 a lysosomal phospholipid salvage pathway that is essential under nutrient
 limitation, and more broadly, provides a robust platform to deorphan
 lysosomal gene function.
description: Endolysosomal library construction A list of
 lysosomal, endocytic and autophagy-related genes in addition to a
 selective set of metabolism and signaling genes (1,061 genes) was manually
 curated by unbiased profiling of lysosomal proteins using in-house LysoIP
 proteomics and by selecting genes implicated in endolysosomal trafficking.
 For the library design, 10 targeting sgRNAs were selected per gene, and an
 additional 1,050 sgRNAs (~9% of the library) were included as
 safe-targeting (control) sgRNAs. These guides were cloned into
 pMCB320. Endolysosomal screen The
 endolysosomal library was infected into Cas9-expressing PaTu-8988T
 pancreatic cancer cells at 1000x library coverage and selected using
 puromycin. At time point 0, cells were trypsinized, washed, and split into
 two culture media conditions: choline-depleted medium (-choline) or
 choline-supplemented medium (+choline). Choline-depleted medium was
 comprised of 1:1 DMEM/Ham’s F12 without sodium bicarbonate, serine,
 methionine, or choline chloride (Caisson Labs; DFP14-5) supplemented with
 1200 mg/L sodium bicarbonate, 18 mg/L methionine, 27 mg/L serine, 5%
 triple-dialyzed FBS and penicillin/streptomycin. To make +choline medium,
 -choline medium was supplemented with 100 μM choline chloride. 
 Cells were propagated in -choline or +choline medium for 14
 doublings while maintaining a library coverage of &gt;1000x. DNA from
 time point 0 (T0) and after fourteen doublings for each condition was
 isolated and sgRNA sequences were PCR-amplified. Illumina universal
 adaptor sequences were subsequently added to sgRNA amplicons by PCR, and
 sgRNA abundances were determined using the Illumina NextSeq 550 with
 NextSeq 500/550 mid-output kit v2.5. Screen
 analysis Sequencing data were analyzed using the
 CasTLE method developed by Morgens et al. In brief, the method calculates
 the most likely maximum effect (phenotype) size (CasTLE effect) among each
 group of gene-targeting sgRNAs by comparing each set to the negative
 sgRNAs (control and safe-targeting) in the library. The method then scores
 the significance of the effect (CasTLE score) by permuting the
 results.