GeCKO v2 library cloning and library virus production

Human CRISPR Knockout Pooled Library A (GeCKO v2, #1000000048) was obtained from Addgene. Library A contains a total of 65,383 sgRNAs (3 sgRNAs for 19,050 genes, 4 sgRNAs for 1864 miRNAs and 1000 non-targeting control sgRNAs). The library virus was produced according to the published protocol. In brief, the library plasmids were electroporated into Stbl3 bacteria (Invitrogen), then transformed bacterial cells were plated on bioassay ampicillin plates for 14-h bacterial culture at 32 °C. The colonies were collected, and the plasmids were isolated and purified using Maxiprep kits (Qiagen). HEK293T cells were transfected with library plasmids, packaging vector psPAX2 and envelope vector pMD2.G. The virus-containing medium was harvested 48–72 h after transfection.

In each independent experiment, we infected ~150 million SUM159 at MOI of 0.3–0.5; corresponding to a cell survival rate of 30–40%. Briefly, 3 million SUM159 cells were plated into each well of 12-well culture plates with 8 µg/ml of polybrene (EMD Millipore Corp. #TR-1003-G). The library virus was added based on the previously optimal titered concentrations allowing for a 30–40% cell survival rate. The plated cells were spin-infected at 1000 × g for 2 h at 32 °C and incubated at 37 °C overnight. Puromycin selection (2 µg/ml) was then performed for 7 days before the cells be divided into three groups. (1) 30 million transduced cells were collected for sequencing to assess library representation. (2) For in vitro drug screen, 40 million infected cells were cultured in T225 flasks in the presence of paclitaxel (10 nM) while another 40 million cells were cultured with vehicle (DMSO) treatment. Cell number counting was performed every 3 days for 2 weeks. (3) For each round of the in vivo screening, 30 million cells/mouse were transplanted subcutaneously into 4 mice. Once tumors were palpable, mice were treated with either paclitaxel (15 mg/kg) or vehicle. Paclitaxel and vehicle were administered once per week over 3 weeks. The mice were then sacrificed, and tumors were snap frozen at −80 °C for subsequent genomic DNA extraction and deep-sequencing.

Genomic DNA was extracted using Qiagen Blood & Tissue Kit (Qiagen) and reference kit protocol was followed. Briefly, 6 ml of NK lysis buffer containing 50 mM Tris, 50 mM EDTA, 1% SDS @ pH 8 and 30 µl of 20 mg/ml Proteinase K (Qiagen) was used for the lysis of 30 million cells or 200 mg of grinded tumor samples. Cells were then incubated for 1 h at 55 °C. Tumors were incubated overnight at 55 °C. Cell lysates were incubated for another 30 min with RNAse A (Qiagen) at the final concentration of 0.05 mg/ml and then placed on ice for 10 min. After adding 2 ml of ice cold 7.5 M ammonium acetate (Sigma), the samples were vortexed and then centrifuged at 4000 × g for 10 min. The supernatants were collected and precipitated by mixing with isopropanol and then centrifuged at 4000 × g for 10 min. The pellets were kept and washed in 70% cold ethanol, air dried and resuspended in 500 µl 1 × TE Buffer at 65 °C for 1 h. The genomic DNA concentration was measured using Nanodrop (Thermo Fisher).

Two-step PCR was performed to prepare the samples for sequencing. The key principle for the first PCR reaction (PCR1) is that the input amount of genomic DNA for each sample must be sufficient to maintain the 300× coverage of the GECKO library. Each sample for sequencing was prepared in PCR1 reactions as follows: 98 °C for 2 min, 98 °C for 10 s, 60 °C for 20 s, 72 °C for 30 s, and 72 °C for 2 min for 18 cycles. Each 100 µl PCR1 reaction contained 20 µl Herculase 5× Buffer, 1 µl of 100 mM dNTP, 2.5 µl of Forward Primer F, 2.5 µl of Reverse Primer, 1 µl Herculase II Fusion Enzyme (Agilent), 10 µg of the extracted DNA and PCR grade water. The adapters specific to Illumina sequencing were attached in the second PCR (PCR2). Each 100 µl PCR2 reaction (20 µl Herculase 5× Buffer, 1 µl of 100 mM dNTP, 2.5 µl of Forward Primer, 2.5 µl of Reverse Primer, 1 µl Herculase II Fusion Enzyme, 5 µl of PCR1 amplicon and 68 µl of PCR grade water) was performed in the same way as PCR1 reaction. The resulting PCR products were run on a 2% agarose gel, then gel extracted and purified using PCR & Gel Cleanup Kit (Qiagen). The library-ready samples were sequenced at Génome Québec (https://www.genomequebec.com/) and 20 million reads capacity was assigned to each sample.

For knockout lentivirus, LentiCRISPR v2 backbone vector was obtained from Addgene (plasmid # 52961). For activation lentivirus, LentiSAM v2 (plasmid #75112) and LentiMPH v2 (plasmid # 89308) were obtained from Addgene. Both knockout and activation sgRNA plasmid cloning procedures followed the Golden Gate cloning protocol [66]. Briefly, the pair of oligo primers for each gene was phosphorylated and annealed in presence of T4 PNK enzyme. Reactions were then incubated at 37 °C for 30 min, 95 °C for 5 min and ramped down to 25 °C at 5 °C/min on a thermal cycler (Bio-Rad). The annealed oligos were diluted 1:10. Golden Gate assembly reaction was performed on the thermal cycler (Bio-Rad). Each reaction contained T7 ligase (Enzymatics), Restriction enzyme (NEB), BSA (NEB), rapid ligase buffer (Enzymatics), annealed oligos, and backbone vector. Each cycle was run at 37 °C for 5 min and 20 °C for 5 min and repeated for a total of 15 cycles. The cloned vectors were further transformed into Stbl3 bacteria (Invitrogen) and seeded on LB agar plates with ampicillin at 33 °C overnight. HEK293T cells were transfected with cloned vector, pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260). After overnight incubation, the culture medium was changed with fresh medium. The supernatant was collected from the culture plates after another 24-h incubation.

All animals were housed and handled in accordance with the approved guidelines of the Canadian Council on Animal Care (CCAC) “Guide to the Care and Use of Experimental Animals”. All experiments were performed under the approved McGill University Animal Care protocol (AUP # 7497 to JJL). All transplantation procedures were undertaken using isoflurane anesthesia. SUM159 cells infected with GECKO library were prepared in PBS (Phosphate Buffered Saline 1X, WISENT INC.) and transplanted into NSG mice by means of subcutaneous injection. For single KO cell transplantation, 1 million SUM159 cells were initially diluted in 20 µl PBS and 20 µl Matrigel (BD Bioscience) and then transplanted into mammary glands of NSG mice. When the tumors became palpable, after 3–4 weeks, paclitaxel (Sigma) and vehicle (control) were intraperitoneally administered twice per week. Paclitaxel was dissolved in 10% DMSO (Sigma), 40% PEG300 (Sigma), 5% Tween-80 (Sigma) and 45% saline. The mice were treated for 2–3 weeks before tumors reached maximum volume of 1000 mm³ and then were euthanized. Tumor volumes were documented. For tail vein injection, 1 million cells were prepared in 100 µl PBS and injected into the median tail vein. The mice were euthanized after ~4 weeks and the lung tissues were collected and stained in Bouin’s solution (Sigma) for at least 48 h. Lung metastatic nodules were counted under a microscope.

Infected SUM159 or MDA-MB-231 cells were seeded into 96-well plates at the density of 5000 cells per well. Cells were treated with DMSO (control) or Paclitaxel (10 nM) after 24 h cell attachment. After 72 h treatment, 7% PrestoBlue Cell Viability Reagent (Invitrogen) was prepared in complete medium and 100 µl of the prepared reagent was added to each well. The cells were incubated at 37 °C for 1 h. Fluorescence was measured using the microplate reader (Tecan) at 535 nm excitation and 615 nm emission.

SUM159 cells were seeded into the ultra-low-attachment 24-well plate at the density of 10,000 cells/well. The culture medium contains HAM’s F12 medium (WISENT INC.), 10 ng/ml EGF (Invitrogen), 10 ng/ml bFGF (Invitrogen) and 1 × B27 (Invitrogen). After 7 days of culture, the number of tumorspheres were counted. Sphere-forming efficiency was calculated as: SFE (%) = number of spheres / number of cells plated × 100%.

Monolayer cells were dissociated into single cells and filtered through a 40 µm cell strainer. In total, 500,000 cells were incubated in prechilled PBS with 2% FBS for half an hour at 4 °C. Cell samples were further incubated with anti-EPCR for 30 min. The non-stained or single-stained samples were used as negative controls. Cells were then washed 3 times with FACS buffer and analyzed with BD FAC SCanto II cytometer (BD Biosciences) and Flowjo software (Tree Star Inc.).

Cells were lysed by TRIzol reagent (Invitrogen), and the total RNA was extracted following the standard procedures. In brief, Reverse Transcription (RT) was performed in each reaction containing RT buffer, 0.1 M DTT, Random hexamers, dNTP, ultrapure water (GIBCO) and M-MLV Reversed Transcriptase (Invitrogen). The real-time PCR was performed with SsoFastTM EvaGreen® Supermix (Bio-Rad) using a RotorGene 6000 PCR thermocycler. The RT-PCR steps are: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 20 s. The paired primers are listed as follows.

GeneArt® Genomic Cleavage Detection Kit (Invitrogen) was used for SURVEYOR assay and detailed procedures were according to the manufacture’s protocol. In brief, cell samples were lysated using cell lysis buffer (2 µl protein grader in 50 µl cell lysis buffer). PRC program (68 °C for 15 min, 95 °C for 10 min and 4 °C for holding) was run for DNA extraction. The PCR amplification was run on the program (95 °C for 10 min, the cycle of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 was repeated 40 times and final extension 72 °C for 7 min). The PCR products was combined with detection reaction buffer. The re-annealing reaction was run on the PCR program (95 °C for 5 min, 95–85 °C at decreasing 2 °C/s, 85–25 °C at decreasing 0.1 °C/s and final holding at 4 °C). Detection enzyme was added to all test samples while water for all the negative control samples. After incubation at 37 °C for 1 h, the products were loaded and run in DNA electrophoresis gel for ~30 min at low voltage. The image was taken by imaging system (Bio-Rad).

The bioinformatic tool, Cutadapt (https://cutadapt.readthedocs.io/en/stable/index.html), was initially used to demultiplex raw FASTQ files. Processed FASTQ files containing only the 20-nucleotide sgRNA sequence were then aligned to the library using MAGeCK count command. MAGeCK robust rank aggregation (RRA) was adopted to analyze abundance change of the sgRNAs and genes.

PRISM drug response and mRNA data were downloaded from DepMap portal (https://depmap.org/portal/). Paclitaxel drug response (EC50) and mRNA profiles of the target genes were extracted from the breast cancer datasets. Integrating EC50 and mRNA data results a file containing 34 genes’ mRNA across 42 breast cancer cells and paclitaxel EC50 (Supplementary File 4). For each gene, correlation was calculated between mRNA and paclitaxel EC50 across breast cancer cell lines.

Statistical analysis was done with GraphPad Prism software 9.0. 0. All in vitro experiments were done at least three independent times. For animal studies, at least five independent animals were used in each condition group. The sample sizes were chosen empirically based on the previous observations. No randomization was needed in the study. If not stated otherwise, all data presented mean ± SD. Each data point represents an individual animal or an independent experiment. If not stated otherwise, the unpaired t-test (two-tailed) was applied to compare the means of two groups. p values < 0.05 were considered significant (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).