Lentiviral human CRISPR pooled genome-wide KO library

The human Brunello CRISPR knockout pooled library was a gift from D. Root and J. Doench (Addgene, 73178)⁵³. In brief, the amplification of the plasmid DNA library was achieved by retransforming 100 µl ElectroMAX Stbl4 electrocompetent cells (Invitrogen, Thermo Fisher Scientific) with 400 ng of the plasmid library in four separate repetitions. For each repetition, the transformation was performed using 25 µl of cells and 100 ng of the library DNA with an ice-cold electroporation cuvette (0.1 cm gap size, Bio-Rad). Cells were shocked at 1.8 kV (preset settings, Ec1; Gene Pulser Xcell Electroporation Systems, Bio-Rad) and immediately mixed with 980 µl prewarmed (30 °C) SOC-medium (NEB). The electroporated cells in SOC-medium (4 ml in total) were combined in a ventilated falcon tube, and 6 ml of additional SOC-medium was added. Next, the 10 ml culture was shaken at 30 °C for 1 h, plated on 12 × 15 cm LB agar plates containing 100 µg ml⁻¹ carbenicillin and incubated overnight for 16 h at 30 °C. The bacteria were next scraped from the plate using a cell scraper after adding 10 ml ice-cold LB medium per plate. The cell suspension was centrifuged (4 × 30 ml) at 4,000 relative centrifugal force (r.c.f.) for 10 min. The plasmid DNA library was extracted from the pellet using the Plasmid Maxi Kit (QIAGEN).

HEK293T cells were seeded in 4× T-225 flasks with 10 × 10⁶ cells per flask in 50 ml medium and incubated for 2 d until reaching 80% confluency. Before transfection, the medium was replaced with fresh medium containing heat-inactivated FBS (56 °C, 30 min). DNA (240 µg in total; 30 µg plasmid library DNA, 20 µg psPAX2 and 10 µg pMD2.G) in 24 ml Opti-MEM I Reduced Serum Medium (Gibco, Thermo Fisher Scientific) was mixed with 720 µl X-tremeGENE HP (Roche) and incubated at room temperature for 20 min. psPAX2 and pMD2.G were gifts from D. Trono (Addgene, 12260 and 12259). Next, this transfection mix was distributed dropwise into the four flasks with HEK293T cells at 80% confluency. Next, 24 h after transfection, the supernatant was collected and replaced with fresh medium containing heat-inactivated FBS. This procedure was repeated for the next 2 d, resulting in a total of 600 ml virus-containing supernatant. The supernatant was centrifuged at 500 r.c.f. for 10 min, and the resulting supernatant was filtered using a syringe filter unit (0.45 µm pore size, polyvinylidene difluoride (PVDF), 33 mm, Millex, Merck) to remove potential remaining cells. The flowthrough was concentrated 10× with Amicon centrifugal units (Ultra-15, PLHK Ultracel-PL Membrane, 100 kDa cut-off, Merck). The concentrated virus supernatant was then aliquoted into 2 ml tubes and frozen at −80 °C. To determine the multiplicity of infection (m.o.i.) per ml supernatant, serial dilutions of the supernatant were performed and combined with 10⁶ HEK293T cells in a total of 3 ml per well on six-well plates (medium with heat-inactivated FBS). Then, 24 h later, the medium was replaced with fresh medium containing 1 µg ml⁻¹ puromycin (Gibco, Thermo Fisher Scientific). Next, 48 h after transduction, the wells were compared to the control well without virus transduction and without puromycin. The wells with survival rates of 10–80% were chosen to determine the m.o.i. per ml of the viral supernatant.

Cells (10⁸) in 500 ml medium with heat-inactivated FBS were combined with lentiviral supernatant at an m.o.i. of ~0.3, corresponding to a ~400× coverage of each sgRNA (library contains 76,441 unique sgRNAs) and were plated on 10× T-225 flasks. Next, 24 h after infection, puromycin was added to a final concentration of 1 µg ml⁻¹. Then, 3 d after transduction, infected cells (cells surviving puromycin selection) were detached using 10 ml per flask Accutase solution (Gibco, Thermo Fisher Scientific). Cells were counted, and ~36 × 10⁶ cells were immediately frozen after pelleting at 500 r.c.f. for 10 min. The remaining cells were replated again on 12× T-225 flasks with 10 × 10⁶ cells per flask in 50 ml. Four flasks were cultured without antibiotics, whereas four flasks were selected with 3 µg ml⁻¹ blasticidin S and another four flasks were selected using 5 µg ml⁻¹ blasticidin S (Gibco, Thermo Fisher Scientific). After 2 weeks of selection (the condition without selection was collected 5 d after it reached 100% confluency), the surviving population of each condition was detached using Accutase, pooled and pelleted at 500 r.c.f. for 10 min. The cell pellets were kept at −20 °C until genomic DNA isolation.

Genomic DNA was isolated from the library-transduced HEK293T EXSISERS_FOXP1:18bBSD cells using the Wizard SV Genomic DNA Purification System (Promega) according to the manufacturer’s protocol. To amplify the CRISPR–Cas9 spacer sequence of the integrated lentivirus, the following barcoded primers with NGS adapters (underlined) were used, comprising an adapter sequence, the barcode (in upper case) and the binding region: BC1: ccatctcatccctgcgtgtctccgactcagCTAAGGTAACggctttatatatcttgtggaaaggacg; BC2: ccatctcatccctgcgtgtctccgactcagTAAGGAGAACggctttatatatcttgtggaaaggacg; BC3: ccatctcatccctgcgtgtctccgactcagAAGAGGATTCggctttatatatcttgtggaaaggacg; BC4: ccatctcatccctgcgtgtctccgactcagTACCAAGATCggctttatatatcttgtggaaaggacg; and a reverse primer composed of the trP1 adapter sequence (underlined) and the binding region: cctctctatgggcagtcggtgatctttcaagacctagctagcgaattc. A primer pair with BC3 was used to amplify genomic DNA from the transduced cells after puromycin selection but before blasticidin S selection. BC1, BC2 and BC4 were used to amplify the condition selected with 0 µg ml⁻¹, 3 µg ml⁻¹ and 5 µg ml⁻¹ blasticidin S. NEBNext Ultra II Q5 Master Mix (NEB) was used to PCR-amplify the spacer sgRNA sequence from the proviral library according to the manufacturer’s protocol. The PCR products were agarose-gel-purified using the Monarch DNA Gel Extraction Kit (NEB). The barcoded PCR products were pooled equimolarly and submitted to the PrimBio Research Institute for NGS analysis. The FASTQ data were presorted according to their barcodes using the software ENCoRE⁶¹. The reads between different pools were normalized to the corresponding pool median before comparison.