The research in this manuscript complies with relevant ethical regulations. The research was approved by Harvard University’s Committee on Microbiological Safety, and animal experiments were approved by the Massachusetts Institute of Technology Institutional Animal Care and Use Committee.

Cell culture and chemicals

Cell lines were from ATCC: K562 (CCL–243), A375 (CRL–1619), C2BBe1 clone of Caco-2 (CRL–2102) and HEK293T (CRL–3216). Cells used in this study were from low-passage cultures from primary stocks, and cell lines were not further authenticated. K562 cells were grown in RPMI (Corning 10–040) supplemented with 10% (v/v) heat-inactivated FBS (Gibco 10438026). HEK293T and A375 cells were grown in Dulbecco’s modified Eagle medium (DMEM, Corning 10–013) supplemented with 10% (v/v) FBS. Caco-2 cells were grown in Eagle’s minimal essential medium (EMEM, ATCC 30–2003) supplemented with 10% (v/v) FBS. All cell lines were grown at 37 °C and 5% CO2, and penicillin and streptomycin were added to all growth media to final concentrations of 100 U ml−1 and 100 μg ml−1, respectively (Corning 30–002–CI). Cells were tested for mycoplasma contamination using the MycoAlert mycoplasma detection kit (Lonza LT07-318).

(1S,3R)-RSL3 (Cayman Chemical 19288), erastin (MedChemExpress HY–15763) and ferrostatin-1 (Selleckchem S7243) were dissolved in dimethyl sulfoxide (DMSO), quality controlled and stored by the ICCB-Longwood screening facility. Fluoxetine HCl (Millipore Sigma F132), serotonin HCl (Tocris 354750) and 5-OH Trp (Millipore Sigma H9772) were prepared in DMSO. For metal ion complementation assays, ZnSO4·7H2O (Fluka 96500), CuSO4 (VWR VW 3312–2), Fe(NO3)3·9H2O (Sigma F–1143) and MnCl2·4H2O (Sigma M8530) were dissolved in H2O to 100 mM. Lactic acid (Fluka 69775) was adjusted to pH 7 with NaOH and diluted with H2O to 0.5 M, and sodium pyruvate (Sigma P2256) and AKB (Sigma K401) were dissolved in H2O to 300 mM and 1 M, respectively.

Transporter CRISPRi/a sgRNA library cloning

Transporter libraries include sgRNAs targeting 413 SLC genes as defined by the Human Genome Organization gene nomenclature, 28 atypical SLCs and 48 ABC transporters (45 as defined by the Human Genome Organization and 3 atypical). sgRNA sequences were from Horlbeck et al.34 and included 10 sgRNAs per gene for all genes, except 37 of them that had 2 transcription start sites and were therefore represented by 20 sgRNAs, and 730 NTC sgRNAs (Supplementary Table 1). The design and cloning of sgRNAs was performed as previously reported34 with minor modifications. A single pool of oligonucleotides (12,000) for both CRISPRi and CRISPRa libraries was synthesized by Twist Biosciences. PCR reactions were set up using primers specific to either CRISPRi or CRISPRa sequences using Phusion polymerase (NEB) according to the manufacturer’s instructions and using three different conditions to minimize amplification bias (HF buffer, GC buffer and GC buffer + 3% DMSO). Each 50 μl reaction included 4.5 ng template and 0.4 μM primers, and was amplified using eight cycles of 30 s at 98 °C, 30 s at 60 °C (57 °C for the DMSO-containing reaction) and 10 s at 70 °C preceded by 1 min at 98 °C and followed by 1 min at 72 °C. Two replicate sets of reactions were run, and all CRISPRi and CRISPRa amplified libraries were pooled separately. The amplified library (88 bp) was purified by agarose gel electrophoresis and extracted using the QIAquick gel extraction kit (Qiagen). Purified PCR products were digested with BlpI and BstXI, and the digested fragment was purified on a 20% polyacrylamide Tris–borate–EDTA (TBE) gel (Thermo Fisher Scientific). Digested fragments were purified by isopropanol precipitation, and DNA concentrations were quantified by fluorescence using the Qubit dsDNA high-sensitivity assay kit (Thermo Fisher Scientific). Plasmid pU6-sgRNA EF1Alpha-puro-T2A-BFP (Addgene #60955) was digested using BlpI and BstXI, purified by agarose gel electrophoresis and extracted using the QIAquick gel extraction kit (Qiagen). Digested PCR fragments were ligated into the restricted vector at a 1:1 molar ratio using T4 DNA ligase and were purified using the MiniElute PCR purification kit (Qiagen). Purified ligated plasmids were electroporated into MegaX DH10B (Thermo Fisher Scientific) and plated on LB/Amp plates. Library coverage was 10,000× for both libraries and was determined by serial dilution and colony counting. After 15 h at 30 °C, the lawn of bacteria was scraped off the plates, and plasmid DNA was prepared using the Plasmid Plus Maxi kit (Qiagen).

Lentivirus preparation

HEK293T cells were transfected with the lentiviral plasmid, psPAX2 (Addgene #12260) and pCMV-VSV-G (Addgene #8454) in a 2:2:1 molar ratio using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. The growth medium was replaced 6 h post-transfection and was then collected at 24–30 h and 48–54 h post-transfection. The two collected growth medium fractions were pooled, centrifuged at 1,000g for 10 min and filtered through a 0.45 μm low-protein-binding membrane. Lentivirus-containing supernatants were stored at −80 °C.

Media preparation

To prepare complete RPMI lacking all amino acids, 8.59 g of RPMI without amino acids, sodium phosphate (US Biological R8999–04 A), 2.00 g sodium bicarbonate (Sigma S6014) and 0.80 g sodium phosphate dibasic (Sigma S0876) were diluted in 945 ml de-ionized H2O. After addition of 100 ml dFBS (Gibco, Thermo Fisher Scientific 26400044) and 10 ml of penicillin–streptomycin 100× solution (Corning 30–002–CI) and homogenization, the medium was sterilized via 0.22 μm membrane filtration (RPMI + dFBS without amino acids). Amino acids were added to this base medium as needed from stock solutions to RPMI levels or to low amino acid screen concentrations, as described in Supplementary Tables 2 and 3. To make complete RPMI used as the control arm in low amino acid screens and experiments, all 19 amino acids were added to complete RPMI without amino acids to RPMI levels (RPMI rich).

Complete RPMI with amino acids present at physiological levels (PAA-RPMI) was prepared from RPMI + dFBS without amino acids as above, except that 965 ml de-ionized H2O was used. To that, amino acids were added to their concentration in human plasma22 using the same stock solutions as for RPMI above. In addition to the amino acids present in RPMI, alanine, cysteine, carnitine, citrulline, creatine, creatinine, N-acetylglycine and ornithine were also added to their level in human plasma. Quantities added are described in Supplementary Table 2. For low amino acid conditions, the corresponding amino acid was added such that the final concentration in PAA-RPMI matched the concentration used in the CRISPRi/a screens.

To prepare RPMI medium for amino acid import assays (RPMI with 16 amino acids present as heavy isotopes), we first added unlabelled cystine, OH-Pro, Met and Trp to RPMI + dFBS without amino acids. This base medium consisting of complete RPMI lacking 16 amino acids was then used to prepare heavy-labelled RPMI and RPMI with low Leu or low Val. Stock solutions of heavy-labelled amino acids (Cambridge Isotope Laboratories) were added to RPMI lacking 16 amino acids to their concentration in RPMI or to the screen concentration (for Leu and Val), as outlined in Supplementary Table 2.

To prepare PAA-RPMI for amino acid import assays, we prepared a complete PAA-RPMI base as above but lacking 16 amino acids. We then added heavy-labelled amino acids from the same stocks as for RPMI to the concentration in PAA-RPMI, as outlined in Supplementary Table 2.

RPMI + FBS and DMEM + FBS were prepared as outlined in ‘Cell culture and chemicals’ section. HPLM (Thermo A4899101) was adjusted with 10% dFBS. ABS (Sigma-Aldrich B9433) thawed at 4 °C was adjusted to 50 μM with cystine·2HCl (Sigma C6727). After 1 h at 37 °C to ensure dissolution, ABS was filtered through a 0.2 μm low-protein-binding membrane.

Amino acid titrations

Single amino acid dropout RPMI medium was prepared for each of the 19 amino acids present in RPMI by adding 18 amino acids to complete RPMI without amino acids. K562 cells grown in complete RPMI were washed 3× using cycles of centrifugation for 5 min at 300g and resuspension of the cell pellet in phosphate-buffered saline (PBS, Corning 21–040 CV). The final cell pellet was first resuspended in PBS at a density of 10 million ml−1 and then added to single amino acid dropout RPMI or complete RPMI to a final density of 0.1 million ml−1. Thirty microlitres of these cell suspensions was pipetted into the wells of 384-well microplates (Thermo Fisher Scientific 164610). Amino acid stock solutions in H2O (as described in Supplementary Table 2) were adjusted to 0.005% Triton X-100 using a 100× stock solution in H2O and were added to wells of the microplates using a D300 digital dispenser (Hewlett-Packard). We confirmed that the highest concentration of Triton X-100 (0.33 parts per million) did not affect proliferation of K562 cells. Each amino acid was tested along a twofold dilution series from 1× to 1/1,024× its concentration in RPMI and including a no amino acid control. Wells on plate edges were filled but not used for any measurements. The drug dispensing arrangement for each amino acid was spatially randomized (and re-organized during data analysis) to minimize bias. Each data point was present in quadruplicates, and three separate identical plates were prepared for viability measurement after 24, 48 and 72 h incubation. Assay plates were incubated at 37 °C/5% CO2 inside containers humidified by sterile wet gauze. After 24, 48 or 72 h, plates were removed from incubation and cooled at room temperature for 10 min, before dispensing 30 μl of CellTiter-Glo (CTG) (Promega) (1:1 dilution in PBS) into each well. Following a 10 min incubation at room temperature, luminescence was measured in a plate reader (BioTek Synergy H1). A control plate with K562 cells dispensed in complete RPMI was prepared and luminescence was monitored at the onset of the experiment (T = 0) and after 24, 48 and 72 h incubation in the same conditions to determine K562 doubling rates in RPMI. Each data point was averaged across four replicates, divided by luminescence at T = 0, was internally normalized within each plate and was displayed on a log2 scale to represent the total number of population doublings.

Preparation of CRISPRi/a parental cell lines

To generate a K562 cell line stably expressing dCas9-KRAB (K562 CRISPRi), K562 cells were transduced with lentiviral particles produced using vector pMH0001 (Addgene #85969; which expresses dCas9-BFP-KRAB from a spleen focus forming virus promoter with an upstream ubiquitous chromatin opening element) in the presence of 8 mg ml−1 polybrene (Sigma). A pure polyclonal population of dCas9-KRAB-expressing cells was generated by two rounds of fluorescence-activated cell sorting (FACS) gated on the top half of BFP-positive cells (BD FACS Aria II) (Extended Data Fig. 8e). In a third round of FACS sorting, single cells from the top half of BFP-positive cells were sorted into microplates to establish monoclonal cell lines. The performance of K562 CRISPRi monoclonal lines in knocking down endogenous genes was evaluated by individually targeting three control genes (ST3GAL4, SEL1L and DPH1) and measuring gene expression changes by RT–qPCR, and the best-performing monoclonal cell line was selected for all work described in this study. The preparation of monoclonal K562 cells expressing CRISPRa machinery (K562 CRISPRa) and of A375 expressing CRISPRi (polyclonal) and CRISPRa (monoclonal) has been described elsewhere25,55.

Preparation of CRISPRi/a cell lines using sgRNAs targeting individual genes and expression analysis by RT–qPCR

Pairs of complementary synthetic oligonucleotides (Integrated DNA Technologies) forming sgRNA protospacers flanked by BstXI and BlpI restriction sites were annealed and ligated into BstXI/BlpI double-digested plasmid pU6-sgRNA EF1Alpha-puro-T2A-BFP (Addgene #60955). Oligonucleotides used to build sgRNAs targeting individual genes are listed in Supplementary Table 4. The sequence of all sgRNA expression vectors was confirmed by Sanger sequencing, and lentiviral particles were produced using these vectors as described above (‘Lentivirus preparation’ section). Parental CRISPRi/a cells were infected with individual sgRNA expression vectors by addition of lentivirus supernatant to the culture medium in the presence of 8 μg ml−1 polybrene. Transduced cells were selected using puromycin (2 μg ml−1 for K562 cells) starting 48 h post-transduction and over the course of 7 days with daily addition of the antibiotic. After 24 h growth in puromycin-free medium, 0.1 million cells were collected and total RNA was extracted using the RNeasy Plus Mini kit (Qiagen). Complementary DNA was synthesized from 0.1–0.5 μg total RNA using Superscript IV reverse transcriptase (Invitrogen) and oligo(dT)20 primers (Invitrogen) following the manufacturer’s instructions. Reactions were diluted two- to fivefold with H2O, and qPCR was performed using PowerUp SYBR Green PCR Master mix (Thermo Fisher Scientific), 2 μl diluted cDNA preparation and 0.4 μM of primers using a QuantStudio 6 Real-Time PCR system (Thermo Fisher Scientific). All qPCR primers are listed in Supplementary Table 4. The expression level of target genes was determined relative to the housekeeping gene GAPDH (log2fold change (FC) versus GAPDH) by subtracting Ct values (ΔCt). Cells were grown in RPMI or in low amino acid medium for 72 h at low confluence with daily media changes before expression profiling.

For Caco-2 cells, we used a one-vector CRISPRi system (Addgene #71236) to create polyclonal KD cell lines. Pairs of complementary synthetic oligonucleotides (Integrated DNA Technologies) forming sgRNA protospacers flanked by BsmBI restriction sites were annealed and ligated into BsmBI digested plasmid pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro. Caco-2 cells were infected using lentiviral supernatant produced from these plasmids. Transduced cells were selected using EMEM containing puromycin at 2–5 μg ml−1 over 12 days. Changes in expression level due to CRISPRi were quantified as above. Cells were grown in RPMI or in low-cystine medium for 72 h at low confluence with daily media changes before expression profiling.

Transporter CRISPRi/a screens in low amino acid medium

Lentiviral supernatant was prepared for both the transporter CRISPRi and CRISPRa sgRNA libraries as described above (‘Lentivirus preparation’ section) and was stored at −80 °C. The multiplicity of infection (MOI) of both preparations was determined by titration onto target cell lines and quantification of the percentage of BFP+ cells 2–3 days post-transduction by flow cytometry (BD Biosciences LSR II).

For transporter CRISPRi/a screens in low amino acid, K562 CRISPRi (or CRISPRa) parental cells (85–100 million) were transduced with lentiviral supernatant at an MOI of 0.25–0.3 in 250 ml culture medium + 8 μg ml−1 polybrene in a 225 cm2 cell culture flask (Costar). Twenty-four hours post-transduction, cells were collected and resuspended in 200 ml fresh medium in 2 × 225 cm2 flasks. Starting 48 h post-transduction, the culture medium was exchanged daily and cells were maintained at 0.5–1.0 million ml−1 in puromycin (1.75 μg ml−1) in 400–500 ml. After 6 days in puromycin, the proportion of BFP+ cells determined by flow cytometry increased to 94–96% of the fraction of viable cells. After recovery for 1 day in puromycin-free medium, 2 × 10 million library cells were collected and stored at −80 °C (T = 0 samples). The remaining cells were grown for 24 h in RPMI/rich (complete RPMI with all amino acids; ‘Media preparation’ section). A total of 120 million cells were collected by centrifugation at 300g for 5 min and washed twice with 25 ml PBS, and the final cell pellet was resuspended in 4.8 ml PBS. Complete RPMI medium with 18 amino acids present at RPMI level and 1 amino acid present at a concentration that limits growth of K562 (‘Media preparation’ section) was prepared, and 50 ml was added to 150 cm2 tissue culture flasks (Falcon 355001) that were preheated and equilibrated to 37 °C/5% CO2. Library cells were added to the flasks to a final density of 0.1 million ml−1. For all amino acids except Trp and Cys, screens were conducted over the course of 16 days with three cycles of 1 day in RPMI/rich and 4 days in RPMI/low amino acid, followed by a final day in RPMI/rich. The growth medium was changed daily on all flasks using centrifugation of a fraction of the culture and resuspension of the pellet into pre-equilibrated growth medium. For medium change after growth in RPMI/rich, cell pellets were washed twice with PBS before resuspension in low-amino-acid medium. The amount of culture centrifuged was adjusted such that the final cell density after medium exchange was 0.1 million ml−1. For Trp and Cys, screens were conducted over the course of 16 days and medium was changed every 36 h (1× 24 h RPMI/rich; 4× pulses of 36 h RPMI/low amino acid; 1× 36 h RPMI/rich; 4× pulses of 36 h RPMI/low amino acid; 1× 36 h RPMI/rich). The RPMI/rich control arm and the low His, low Arg and low Lys were run in technical replicates. At the end of the screen, 12–15 million cells were collected by centrifugation, washed once with PBS and stored at −80 °C. The number of population doublings for each screen is indicated in Supplementary Table 3. Genomic DNA (gDNA) was extracted from cell pellets using the QIAamp DNA Blood Mini Kit according to the manufacturer’s instruction, except that the elution was performed using 10 mM Tris HCl pH 8.5. Typical yields from 15 million cells ranged from 90 to 240 μg gDNA. sgRNA barcodes were amplified by PCR using the gDNA from at least 6 million cells as template and Phusion (NEB M0530) as polymerase. An equimolar mix of primers with stagger regions of different length (CC_LSP_025 to CC_LSP_032_c) was used as forward primer, and barcoded index primers (CC_Cri_a_rev1 to CC_Cri_a_rev10) were used as reverse primers. Reactions were composed of 1× HF buffer, 0.2 mM dNTPs, 0.4 μM forward primer mix, 0.4 μM indexed reverse primer, 0.5 μl Phusion, 1.5 mM MgCl2 and 5 μg gDNA in a volume of 50 μl. After 30 s at 98 °C, the reactions were subjected to 23 cycles of 98 °C for 30 s, 62 °C for 30 s and 72 °C for 30 s, and were followed by 72 °C for 5 min. All reactions from each screen were pooled, and the amplified PCR product (~240–250 bp) was purified by agarose gel electrophoresis using the QIAquick gel extraction kit (Qiagen). Purified PCR products were quantified by fluorescence using the Qubit dsDNA high-sensitivity assay kit (Thermo Fisher Scientific). Individual indexed libraries were mixed in equimolar ratio and were further purified using a QIAquick PCR purification kit (Qiagen). After quantification by qPCR using the NEBnext library quant kit for Illumina (NEB), pooled libraries were sequenced on an Illumina HiSeq 2500 platform using a 50 bp single read on a high-output standard v4 flow cell with a 10–20% PhiX spike-in. About 15 million reads were obtained for each indexed screen.

For some of the screens, an alternative strategy was used to accommodate sequencing on an Illumina NextSeq 500 platform. Changes to the protocol above include amplification of extracted gDNA by PCR using a barcoded forward primer (CC_fwd1 to CC_fwd20) and a barcoded reverse primer (CC_Cri_a_rev1 to CC_Cri_a_rev20) using Q5 polymerase (NEB M0491L). Pooled libraries were sequenced on an Illumina NextSeq 500 platform using a 75 bp single read on a high-output flow cell with a 2–5% PhiX spike-in. A total of 15–30 million reads were obtained for each indexed screen.

Sequencing data were analysed as previously reported25 with the following modifications. Trimmed sequences were aligned to the library of protospacers present in the transporter CRISPRi/a sgRNA libraries (Supplementary Table 1), and 89–92% of the number of raw reads typically aligned to the library of protospacers. To estimate technical noise in the screen, simulated negative control genes (the same number as that of real genes) were generated by randomly grouping 10 sgRNAs from the pool of 730 NTC sgRNAs present in the libraries. For each gene (and simulated control gene), which is targeted by ten sgRNAs, two metrics were calculated: (1) the mean of the strongest 7 rho phenotypes by absolute value (‘phenotype score’) and (2) the P value of all 10 rho phenotypes compared with the 730 NTC sgRNAs (Mann–Whitney test). To display data compactly, we calculated a single score (‘screen score’) by multiplication of the phenotype score with −log10(P value). sgRNAs were required to have a minimum of 100 counts in at least one of the two conditions tested to be included in the analysis. To deal with the noise associated with potential low count numbers, a pseudocount of 10 was added to all counts. Gene-level phenotype scores and P values are available in Supplementary Tables 5 and 6.

For screens in low amino acid conditions, scores were calculated using two technical replicates for Arg, Lys and Val. For all other conditions, scores were calculated using one replicate for the low amino acid condition and two technical replicates for both T0 and untreated samples. The highest-scoring negative control gene in any condition was used as the significance cut-off for all conditions (for CRISPRa, a significant hit had a screen score >0.425 or <−0.45; for CRISPRi, >0.49 or <−0.40). We excluded significant hits that had single scores <−0.12 in the untreated control arm of the screen unless they were either hypersensitive or very strong resistant (>1.5) hits in any of the low amino acid conditions. This exclusion was performed because a slowdown in proliferation rate leads to pan-resistance across screen conditions, as previously reported25.

For in vivo screens, scores were determined without normalization to the number of population doublings. The number of sgRNAs required in at least one of two conditions tested was lowered to 25. For comparison with in vitro screens, screen scores were normalized to the most hypersensitive hit (set to −1) in a given screen. Significance cut-offs were determined by the highest-scoring pseudo negative gene (K562 CRISPRi, <−0.075 and >0.025; K562 CRISPRa, <−0.15 (−0.20 for subcutaneous) and >0.05 (0.10 for subcutaneous and ABS); A375 CRISPRi, <−0.05 and >0.03 (0.06 for subcutaneous); A375 CRISPRa, <−0.17 (−0.25 for subcutaneous) and >0.1 (0.7 for subcutaneous).

Phenotype similarity across environments was determined using all genes with a significant deleterious or beneficial phenotype in any of the environments tested. Pairwise Pearson correlations (and P values) were calculated using the function rcorr in the R package Hmisc (v. 4.7.1), and data were displayed using the R package corrplot (v. 0.84).

For CRISPRi screens in K562 in RPMI, two biological replicates each consisting of two technical replicates were collected. To compute a volcano plot of the data, the read counts of the two technical replicates were averaged after normalization to the total number of reads and the resulting two replicates were processed in the analysis pipeline, as explained above. Essential transporters were determined by averaging screen scores determined for each biological replicate. A stringent significance threshold was determined by the highest-scoring pseudogene in any of the two replicates before averaging (score <−0.13). Essential transporters were arranged in a chord plot using GOPlot (v. 1.0.2).

For K562 CRISPRi screens in different culture conditions, significance cut-offs for each screen were determined by the highest-scoring pseudogene. Genes that were significant in at least one condition were displayed in tile plots. However, genes that did not have a growth score <−0.20 or >0.10 in any condition were excluded from the plots. For the comparison between K562 and A375 transporter essentiality, phenotype scores were used instead of growth scores to account for potential differences in sgRNA efficacy. Genes having a phenotype score <−0.065 were significant and were displayed in the tile plot.

Transporter CRISPRi/a screens in rich medium

For K562 CRISPRi/a transporter screens in non-growth-limited conditions, pooled libraries were prepared as above. Cells were collected by centrifugation at 300g for 5 min, washed in PBS and resuspended in PBS at 100 million ml−1. Screens were initiated by addition of 7.5 million cells into 50 ml medium in 150 cm2 flasks and were conducted by passaging the libraries in growth medium for 14 days with medium exchange at least each 2 days and keeping the density between 0.1 million ml−1 and 0.4 million ml−1. The pools of cells were collected at T = 14 days and T = 0 days, and enrichment was analysed as outlined above. For RPMI + 10% FBS at high cell density, cells were kept at a density of 0.3–1.2 million ml−1 with medium exchange every 2 days.

For A375 screens, CRISPRi/a parental cells grown in RPMI + 10% FBS in 4× 15 cm cell culture dishes (Falcon 353025) to 80–90% confluence were transduced with transporter library lentiviral supernatant at an MOI of 0.25–0.3 in presence of 8 μg ml−1 polybrene. Starting 48 h post-transduction, cells were passaged daily to maintain <95% confluence in RPMI + 10% FBS + puromycin (1.0 μg ml−1). After 4 days in puromycin, the proportion of BFP+ cells determined by flow cytometry increased to 90–95% of the fraction of viable cells. After recovery for 1 day in puromycin-free medium, library cells were trypsinized and then quenched. Cells were collected by centrifugation at 300g for 5 min, washed in PBS and resuspended in PBS at 100 million ml−1. Screens were initiated by addition of 15 million cells into 50 ml medium in 15 cm dishes and were conducted by passaging the libraries in growth medium for 14 days with medium exchange at least every 2 days and keeping the confluence between 20% and 80%. Cells were collected at T = 14 days and T = 0 days, and enrichment was analysed as outlined above.

Transporter screens in subcutaneous tumours in immunodeficient mice

All animal experiments conducted in this study were approved by the MIT Institutional Animal Care and Use Committee. A maximum tumour burden of 2 cm was permitted per Institutional Animal Care and Use Committee protocol, and these limits were not exceeded. Male mice between 3 and 4 months old were used in this study. All animals were housed at ambient temperature and humidity (18–23 °C, 40–60% humidity) with a 12 h light and 12 h dark cycle and co-housed with littermates with ad libitum access to water. For animal injections, pooled cells were washed twice with PBS and filtered through a 40 μm cell strainer. After centrifugation at 300g for 5 min, pelleted cells were resuspended at 100 million ml−1 in PBS and the suspensions were kept on ice until injection (<1 h). NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice (NSG mice; The Jackson Laboratory, strain #005557) were injected subcutaneously into both flanks with 0.1 ml of pooled cell suspension (10 million cells per tumour). Each screen was conducted with three mice (N = 6 tumours) over 14 days. At the endpoint, animals were killed and whole tumours (typically 100–500 mg) were excised and kept at −80 °C. gDNA was extracted from homogenized whole tumours (at least 160 μg for each tumour) using the DNeasy Tissue kit (Qiagen). sgRNA barcodes were amplified by PCR using 160 μg of gDNA, as described above for transporter screens in low amino acid screens. Libraries were quantified, pooled and analysed as described earlier. For each screen, the two samples that had the greatest number of NTC counts not within 1 log2 of the median were excluded from the analysis to reduce technical noise. Remaining replicates were paired and read counts were averaged after correcting to match total read counts. These averaged samples were then processed with the T = 0 days samples to determine gene-level scores as outlined above.

Determination of engraftment frequency in subcutaneous tumours in NSG mice

Lentivirus was prepared from plasmid pLJM1–eGFP (Addgene #19319). K562 CRISPRi + transporter library cells were infected with pLJM1–eGFP lentivirus, and the proportion of GFP+ cells was determined by flow cytometry 72 h post-transduction. GFP+ cells were spiked into K562 CRISPRi + transporter library to a final ratio of 1 to 1 × 103, 1 to 1 × 104 or 1 to 1 × 105. For each dilution and unspiked control, cells were prepared as above, diluted with PBS, and 100 μl (0.1, 1 or 10 million cells) was injected into both flanks of NSG mice. In addition, a portion of each preparation was collected for T = 0 days samples and for growth in RPMI + FBS. Tumours were allowed to form over the course of 19 (10 million cells injected), 26 (1 million) and 34 days (0.1 million). At the endpoint, animals were killed and whole tumours were excised and kept on ice. Tumours were dissociated, and the presence of GFP+ cells was determined by flow cytometry analysis. To determine the skewness of the NTC sgRNA distribution, tumours were analysed in the same way as the screens.

Immunoblotting

RPMI medium containing low amino acid was made in the same way as for CRISPRi/a screens. In addition, RPMI containing no Arg, His or Lys was prepared analogously, and we used RPMI/rich and RPMI without amino acids as controls. For the time course, K562 cells growing in RPMI/rich were collected, washed twice with treatment medium, and resuspended in treatment medium at 0.35–0.45 million ml−1. After 24 h, cells were pelleted and resuspended in fresh medium at 0.35–0.45 million ml−1. For each timepoint, 0.6 million K562 cells were pelleted by centrifugation at 300g for 3 min and flash frozen in liquid nitrogen. Pellets were lysed in 1× RIPA buffer (50 mM Tris HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate and 1% NP40) and lysates clarified by centrifugation at 21,000g for 10 min at 4 °C. Protein concentration in lysates was quantified by bicinchoninic acid assay (Pierce). Then, 20–25 μg protein was run on a 4–20% Bis-Tris Bolt gel (NuPAGE) in 1× MES buffer (NuPAGE) at 125 V for 90 min, and transferred to 0.45 μm nitrocellulose at 18 V for 60 min using the Trans-Blot SD semi-dry transfer system (Bio-Rad) in Tris–glycine transfer buffer (10 mM Tris base, 0.1 M glycine and 20% MeOH). The membrane was blocked with 5% skimmed milk or bovine serum albumin (for phospho-antibodies) in TBST for 1 h at room temperature, and incubated overnight at 4 °C with primary antibody in the same solution used for blocking. After 3× 5 min washes with TBST, incubation for 1–3 h at room temperature with 1:5,000 secondary antibody (Cell Signaling Technology goat HRP-linked anti-rabbit IgG #7074), and three more 5 min washes with TBST, blots were developed in 2 ml Western Lighting Plus chemiluminescent substrate (PerkinElmer) and imaged on the ImageQuant LAS4000 (Cytiva). After imaging, the blot was incubated for 15 min in Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) followed by 1 h in 30% H2O2 at 37 °C. After extensive washes with TBST, the blot was blocked and reprobed as above. For each experiment, a single blot was serially assayed with the following rabbit antibodies (Cell Signaling Technology): vinculin (E1E9V) #13901 dilution 1:2,000; phospho-p70 S6 kinase (Thr389) #9234 dilution 1:1,000; phospho-eIF2α (Ser51) (D9G8) #3398 dilution 1:1,000; phospho-4E-BP1 (Ser65) #9451 dilution 1:1,000; p70 S6 kinase #9202 dilution 1:1,000; eIF2α #9722 dilution 1:2,000; 4E-BP1 (53H11) #9644 dilution 1:2,000.

For plasma membrane fractions, we prepared a fresh 10 mM solution of EZ-link-sulfo-NHS-SS-biotin reagent (Thermo Fisher Scientific A39258) in H2O. Cells were grown in low amino acid media for 72 h at low confluence with daily media changes or in regular RPMI. Cells (5–20 million) were washed twice with ice-cold PBS and resuspended to 40 million ml−1 in PBS. EZ-link-sulfo-NHS-SS-biotin was added to 800 μM, and cells were incubated for 30 min at room temperature with gentle rocking. Cells were pelleted and resuspended in 500 μl 150 mM glycine and incubated for 2 min. After washing with 1 ml PBS, cell pellets were resuspended in lysis buffer (50 mM Tris HCl pH 7.5, 100 mM NaCl, 5 mM EDTA pH 8, 1% Triton X-100, 1× protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific 1861280)) at 22.2 μl per 1 million cells. Cells were lysed at 4 °C for 10 min, and lysates were clarified by centrifugation at 21,000g for 10 min at 4 °C. Supernatants (‘total extracts’; about 3.6 μg μl−1) were stored at −80 °C. For western blots, 1/6 volume of 6× Laemmli sample buffer was added and samples were incubated at 95 °C for 5 min. For pull-downs, 100–300 μl total protein extract was added to a tube containing 25 μl of streptavidin agarose resin (Thermo Fisher Scientific 20357) washed once with lysis buffer. After rotating samples at 4 °C for 60 min, the resin was washed with 3× 500 μl lysis buffer and centrifugation at 500g for 3 min. Biotinylated proteins were eluted by incubating washed beads with 1× Laemmli sample buffer (1 μl per 7.5 μg input total protein) at 95 °C for 5 min.

For plasma membrane fraction western blotting, 10–30 μg total protein extract and the equivalent of 30–90 μg input protein of pull-down samples were run on 4–15% Mini Protean TGX precast gels (Bio-Rad) and transferred to nitrocellulose using the iBlot3 system and reagents (Invitrogen) at 25 V for 3 min. Membranes were blocked in Intercept (TBS) Blocking Buffer (LI-COR) for 1–2 h at room temperature, and then incubated overnight at 4 °C with primary antibody in blocking buffer + 0.2 % Tween-20 (SLC7A5 (Proteintech, 28670-1-AP) 1:5,000; SLC3A2/4F2hc (Cell Signaling, D603P, #13180) 1:1,000; SLC7A6 (Novus Biologicals, NBP2-75086) 1:500; SLC7A7 (Novus Biologicals, NBP1-82826) 1:500; SLC2A1 (Cell Signaling, D3J3A, #12939) 1:1,000). After 3× 5 min washes with TBST, incubation for 1–3 h at room temperature with 1:20,000 anti-rabbit secondary antibody (IRDye800CW LI-COR 926-32211) or 1:10,000 anti-goat secondary antibody for SLC7A6 only (donkey IgG H&L, Alexa Fluor 750 conjugate, Abcam ab175744) in blocking buffer + 0.2% Tween-20 + 0.01% sodium dodecyl sulfate. After 3× 5 min washes with TBST, blots were imaged on the iBright FL1500 Imaging System (Invitrogen). For vinculin staining post-imaging, blots were re-processed as above starting with an overnight incubation in 1:2,000 vinculin (Cell Signaling, E1E9V, #13901) in blocking buffer + 0.2 % Tween-20.

Growth competition assays

Growth phenotypes were measured in competition assays where a test cell line was mixed with its corresponding NTC control at a 1:1 ratio. Cell lines were grown in complete RPMI, and cell densities were determined using a TC20 automated cell counter (Bio-Rad). Cell mixes were prepared by combining 1 million test cells with 1 million NTC cells, washing twice with 10 ml PBS, and resuspending the final pellet in 1 ml PBS. Fifty microlitres of resuspended cells (0.1 million) was added to wells of a 12-well plate containing 1 ml of growth medium, and an aliquot was stored at −80 °C to determine the initial ratio for each mix. Cultures were passaged as necessary (that is, daily for low amino acid screen validation) by centrifugation of a portion of the culture and resuspension in fresh medium. At the end of the experiment, the total number of population doublings was determined for all conditions and 0.1–0.2 million cells were collected by centrifugation. gDNA was extracted from cell pellets using the QIAamp DNA Blood Mini Kit according to the manufacturer’s instruction, except that the elution was performed using 10 mM Tris HCl pH 8.5 (typical yields were 2–10 μg). Each extracted gDNA sample was assessed in two qPCR reactions using either a forward primer complementary to the test cell sgRNA sequence or to the NTC sgRNA sequence, and a universal reverse primer. Primers were tested beforehand to ensure linearity and specificity of detection. The composition of the mix was estimated by the difference in Ct value between the two reactions (ΔCt). Growth phenotypes were determined by comparing the ΔCt post-growth in the test medium with the ΔCt of the sample taken at T = 0 (ΔΔCt), and then normalizing by the number of population doubling differences between the test medium and a vehicle control (for example, low amino acid versus RPMI/rich). To determine growth phenotypes in complete media, ΔΔCt values were normalized by the number of population doublings between T = 0 and the end of the assay.

Amino acid transport assays in K562 cells

The polymer coverslip in a 35 mm µ-Dish (Ibidi 81156) was coated with Cell-Tak. For each dish, 12.25 μg Cell-Tak (Corning 354240) diluted to 360 μl with H2O was neutralized with 40 μl 1 M bicarbonate pH8 solution and rapidly spread over the surface of the coverslip. After 45 min at room temperature, the solution was removed and the coverslip was thoroughly washed with 2× 800 μl H2O and subsequently left to dry for a few minutes. To prepare cells for immobilization, K562 were collected (1 million cells per dish), washed once with RPMI without FBS and resuspended in RPMI without FBS (400 μl per dish). Resuspended cells were added to coated dishes and incubated at room temperature for 30–45 min. Medium and unattached cells (typically, 0.8 million cells are needed to form a homogeneous monolayer of K562 cells) were gently removed by aspiration. After one wash with 500 μl RPMI/rich, cells were incubated for at least 2 h at 37 °C/5% CO2 in 500 μl RPMI/rich to reach steady state.

For import assays, one dish is required for each timepoint for each cell line. We typically assayed amino acid import over seven to eight timepoints (0, 5, 10, 20, 30, 40, 100 and 250 s) to capture the initial slope for all 16 amino acids. RPMI/rich was removed by aspiration and was replaced by 400 μl pre-warmed and pre-equilibrated RPMI + 16 heavy-labelled amino acids. After incubation at 37 °C for the duration of the timepoint (on a heat block for short timepoints and in the incubator for longer timepoints), cells were thoroughly washed by sequentially submerging dishes into 4× 400–600 ml ice-cold PBS. For the 0 s timepoint, the same procedure as above was performed but using RPMI/rich instead of heavy-labelled medium and incubating for 10 s. PBS in the washes was refreshed regularly to ensure limited contamination of intracellular amino acid pools. After the last wash, PBS was removed by aspiration and 600 μl of ice-cold extraction buffer (80% MeOH/20% H2O spiked with 4 μg ml−1 norvaline (Sigma N7627)) was added. Cells were scraped off the plate on ice (United Biosystems MCS–200) and were transferred to 1.5 ml tubes. Samples were homogenized in a thermomixer at 2,000 rpm and 4 °C for 15 min. Tubes were spun at 21,100g for 10 min at 4 °C, and supernatants were transferred to new tubes containing 10 μl of 0.1 N HCl and were stored at −80 °C until analysis by GC–MS.

For amino acid consumption assays, a single dish was required for all timepoints for each cell line. We typically assayed consumption over five timepoints (0, 1, 2, 3 and 4 h) in RPMI and over four timepoints (0, 5, 15 and 60 min) in low amino acid medium. RPMI/rich was removed by aspiration and was replaced by 300 μl pre-warmed and pre-equilibrated RPMI/rich. At each timepoint, 15 μl of medium was removed and kept on ice. All samples were spun at 1,000g for 5 min at 4 °C, and 10 μl of the supernatant was transferred to a tube containing 600 μl of extraction buffer + 10 μl of 0.1 N HCl. Samples were stored at −80 °C until analysis by GC–MS.

For GC–MS analysis, samples were dried at room temperature using a flow of nitrogen. To each tube, we added 24 μl methoxamine reagent (ThermoFisher TS–45950) and incubated the resuspended samples at 37 °C for 1 h. We next added 30 μl N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide + 1% tert-butyldimethylchlorosilane (Sigma 375934) and incubated homogenized samples at 80 °C for 2 h. After centrifugation at 21,100g for 10 min, supernatants were transferred to glass vials. Derivatized samples were analysed on a DB-35MS column (Agilent Technologies) in an Agilent 7890B gas chromatograph linked to an Agilent 5977B mass spectrometer. One microlitre of sample was injected at 280 °C and mixed with helium carrier gas at a flow rate of 1.2 ml min−1. After injection, the oven was held at 100 °C for 1 min and ramped to 250 °C at 3.5 °C min−1. The oven was then ramped to 320 °C at 20 °C min−1 and held for 3 min at 320 °C. Electron impact ionization in the mass spectrometer was performed at 70 eV, and the MS source and quadrupole was held at 230 °C and 150 °C, respectively. Scanning mode was used for detection with a scanned ion range of 100–650 m/z. Acetone washes were performed after every three to five samples and before and after each run. GC–MS raw data were quantified using El-MAVEN (v0.11.0), and, for each amino acid, we extracted the area of the peak for the fragment with the highest signal over noise for the parent ion and all detectable isotopologues (fragments are highlighted in Fig. 2b). Natural isotope abundance was subsequently corrected using IsoCorrectoR to determine total ion counts for each amino acid fragment70. For import assays, each sample was first normalized using the norvaline internal standard. We corrected each sample by a factor determined by calculating the ratio of the norvaline signal to the average of the norvaline signal over all timepoints. As the experiment was run under steady state conditions, we applied a second correction that took advantage of the fact that the total ion count for all amino acid fragments (labelled and unlabelled) remains constant over all timepoints. We calculated the total ion count (excluding norvaline) for each sample and corrected each sample by a factor determined by calculating the ratio of the total ion count for the sample to the average total ion count over all timepoints. To determine absolute amounts in each sample, we used a standard mix (Cambridge Isotope Laboratories MSK–A2) containing 17 amino acids to which we added Gln and Asn. We made dilutions of this mix in 0.1 N HCl and added them to 600 μl K562 cell extract prepared as for the import assay. We analysed these samples as above and applied a linear regression to the data. We then used the linear regression data and the mean norvaline signals of the standard and the sample to convert ion counts into pmol amino acid. Data were normalized by the number of cells used in the assay (0.8 million in cell monolayer minus an estimated 20% cell loss during the washing steps). Intracellular amino acid levels were calculated by averaging the sum of unlabelled and heavy-labelled amino acid over all the timepoints. To calculate import rates, we determined the initial slope of the increase in intracellular levels of heavy-labelled amino acids. We applied a linear regression to heavy-labelled amino acid levels over time, and the number of data points included was variable depending on the amino acid and the conditions. We typically used data from 0 to 100 s for Asn, Asp, Gln, Glu and Pro; data from 0 to 40 s for Arg, Gly, His, Ile, Lys, Ser, Thr and Val; data from 0 to 20 s for Leu and Tyr; and data from 0 to 10 s for Phe.

For import assays in low-arginine and low-lysine conditions (and RPMI control), we used liquid chromatography–mass spectrometry (LC–MS) to quantify light- and heavy-labelled amino acids in methanol cell extracts prepared in the same way as for GC–MS. Samples were dried down using a flow of nitrogen, resuspended in 100 μl 80% MeOH/20% H2O and filtered through a 10 kDa PES filter. LC–MS analysis was performed using a QExactive orbitrap mass spectrometer using an Ion Max source and heated electro-spray ionization probe coupled to a Dionex Ultimate 3000 UPLC system (Thermofisher). For this, 2.5 μl sample was injected onto a SeQuant ZIC-pHILIC 2.1 mm × 150 mm (5 μm particle size) column (Millipore Sigma). The flow rate was set to 0.15 ml min−1, and temperatures were set to 30 °C for the column compartment and 4 °C for the autosampler tray. The following conditions were used to achieve chromatographic separation: mobile phase A was 20 mM ammonium carbonate, 0.1% ammonium hydroxide, and mobile phase B was 100% acetonitrile. The chromatographic gradient was as follows: 0–20 min, linear gradient from 80% to 20% B; 20–20.5 min, linear gradient from 20% to 80% B; 20.5–28 min, hold at 80% B. The mass spectrometer was operated in full scan, polarity-switching mode, the spray voltage was set to 4.2 kV and the heated capillary was held at 320 °C. The MS data acquisition was performed in a range of 70–1,000 m/z, with the resolution set at 70,000, the AGC target at 1 × 106 and the maximum injection time at 20 ms. Quantification of light- and heavy-labelled amino acid levels from LC–MS raw data and import rates were determined as for GC–MS without natural abundance correction and calculation of absolute levels.

For consumption experiments, each sample was normalized using the norvaline standard as above. We next adjusted sample signals to account for the decrease in volume of medium at each timepoint. Finally, we made use of the fact that the experiment was run under steady-state conditions to correct samples on the basis of the total ion count (excluding norvaline) of each sample. We applied a linear regression to the total ion count over the time course of each experiment. We then corrected each sample by a factor calculated by taking the ratio of the total ion count of the sample to the computed level determined by the regression. To determine absolute amounts in each sample, we diluted 10 μl of the standard mix prepared above into 600 μl extraction buffer + 10 μl RPMI/rich and analysed these standards as detailed for the samples above. We applied a linear regression to the standard data and converted sample ion counts to pmol using the regression data as well as the mean norvaline signals of the standards and samples. Consumption rates were calculated from the slope of the changes in amino acid levels over the time course of the assay determined by linear regression.

Amino acid export was measured by exposing cells to RPMI containing heavy-isotope-labelled amino acids, rapidly switching to regular RPMI and measuring the release of labelled amino acids into the medium. While this approach was technically challenging due to the rapidity of export and the effects of differences in intracellular amino acid levels on export rates, we were able to quantify export rates for a subset of amino acids. K562 cells attached in a monolayer on the surface of an Ibidi dish were prepared as for the import assay. Cells were incubated for at least 2 h with RPMI containing heavy-labelled amino acids at 37 °C/5% CO2. Growth medium was removed by aspiration, and cells were thoroughly washed by sequentially submerging dishes into 4× 400–600 ml PBS at room temperature. After aspiration of the last PBS wash, 160 μl pre-warmed and pre-equilibrated RPMI/rich was added to the dish. Plates were kept at 37 °C, and the medium was homogenized by regularly swirling the dishes. Samples of the media (12.5 μl) were taken over the course of the assay (0, 5, 10, 20, 30, 40 and 100 s) and kept on ice. Samples were centrifuged at 300g for 5 min at 4 °C, and 10 μl of the supernatant was transferred to a tube containing 600 μl of extraction buffer + 10 μl of 0.1 N HCl. Samples were stored at −80 °C until analysis by GC–MS that was performed and analysed in the same way as the consumption samples.

Cell viability assays

For K562, cells growing in RPMI/10% FBS were collected by centrifugation, washed 2× with PBS and resuspended at 10 million ml−1 in PBS. Medium was prepared for each growth condition by addition of small molecules to RPMI low cystine using DMSO stocks. K562 cells were added to each medium to a final concentration of 0.1 million ml−1, and suspensions were dispensed into wells of a 48-well plate in triplicates. After 72 h incubation at 37 °C/5% CO2, 50 μl of each culture was transferred to a 96-well plate (in duplicates), 50 μl CTG reagent was added and luminescence was measured in a plate reader (BioTek Synergy H1). Viability was quantified by normalization to luminescence at T = 0 h. For the cystine titration, K562 cells washed in PBS were resuspended in RPMI −Cys (0.1 million ml−1) and dispensed in a 96-well plate. Cystine was added using a D300 digital drug dispenser (HP), and viability at 72 h was determined by addition of CTG reagent and measurement of luminescence.

For A375, cells were trypsinized and resuspended in RPMI −Cys, then washed 2× with RPMI −Cys and finally resuspended at 10 million ml−1 in the same medium. Cells were added to growth medium to 0.1 million ml−1 and dispensed into wells of a 96-well plate. After 2 h at 37 °C/5% CO2, small molecules were added to wells using a D300 digital drug dispenser (HP) using stocks in DMSO. Viability was determined at T = 48 h by addition of CTG reagent and measurement of luminescence.

For Caco-2 assays, 96-well microplates were coated with poly-d-lysine to avoid cell loss during washing steps. Forty microlitres of a 1:1 (v/v) solution of poly-d-lysine (Sigma A–003–M) 0.1 mg ml−1 in H2O and PBS was added to each well and incubated for 1 h at room temperature. Wells were washed 3× with PBS and left to air dry. Caco-2 cells were seeded into the 96-well plates in EMEM/10% FBS at a density of about 1,000 cells per well. After 48 h at 37 °C/5% CO2, wells were washed twice with RPMI −Cys followed by addition of 50 μl RPMI −Cys. Small molecules were added to wells using a D300 digital drug dispenser (HP). Viability was quantified at T = 48 h using the CTG luminescence assay and compared with viability at T = 0 h.

Lipid peroxidation quantification

K562 cell lines growing in RPMI/rich were collected, washed 2× with PBS and incubated for 24 h in RPMI −Cys at 1 million ml−1. After media exchange to fresh RPMI −Cys and dilution to 0.5 million ml−1, cells were dispensed in a 12-well plate (2 ml per well). Cystine was added from a 200× aqueous stock solution, and RSL3 and serotonin were added using 1,000× stocks in DMSO. After 6 h incubation at 37 °C/5% CO2, 100 μl of a solution of BODIPY 581/591 C11 (Thermo Fisher D3861) 200 μM in RPMI −Cys (5% v/v DMSO) was added to each well. After 30 min incubation at 37 °C/5% CO2, cells were washed once with PBS and analysed by flow cytometry (BD LSRFortessa). The oxidized form of BODIPY C11 was quantified using 488 nm emission and 530/30 nm emission, and the reduced form was quantified using 561 nm emission and 610/20 nm emission. Peroxide levels were estimated by the ratio of these two fluorescence intensities.

Statistics and reproducibility

All experimental assays, excluding CRISPR screens, were performed at least in triplicate. The exact number of replicates is indicated in the figure legends. In general, data represent the mean of individual replicates and error bars represent the standard error of the mean (s.e.m.). Data analysis and display was accomplished in R (v. 3.6.0) using ggplot (v. 3.3.3). Significance probabilities, except for CRISPR screens, were calculated using two-tailed unpaired Student’s t-tests. For western blots, experiments were reproduced twice with similar results and one of the replicates was shown as representative example. No sample size calculation was performed for experiments in cell culture. For in vivo screens, we pre-determined in initial experiments that sufficient library representation was achieved in every single tumour and we included six replicate tumours for each screen condition. To improve data quality in these screens, we excluded from each set of screens the two datasets with the highest amount of technical noise. Noise was determined in an unbiased way by quantifying the number of NTC sgRNA counts that were within 1 log2 of the median after growth in vivo. No other data were excluded from the study. Transporter CRISPR screens were run in duplicates, and all attempts at replication were successful (Extended Data Fig. 2c,d). Mice were randomly chosen for the four subcutaneous injection conditions. Other experiments were not randomized as there was no allocation into different experimental groups. The researcher performing subcutaneous injections was blinded to the identity of the cells injected. All pooled CRISPR screens are inherently randomized and blinded. Other experiments were not blinded as all data were collected using unbiased methods.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.