CRISPR editing of primary human NK cells using retroviral sgRNA transfer and Cas9 electroporation
Primary human NK cells were transduced on day 5 post-isolation using the retroviral sgRNA transfer plasmid containing a specific sequence targeting PTPRC (CD45). Following transduction, NK cells were electroporated on d7 using the Lonza 4D nucleofector X unit (Cat #AAF-1003X). NK cells were washed with PBS, pelleted, and resuspended in P3 primary buffer, supplemented with Cas9 (10.16µM; Alt-R™ S.p. Cas9 Nuclease V3, Integrated DNA Technologies, Cat# 10000735) and electroporation enhancer (4.16µM; Alt- R™ Cas9 Electroporation Enhancer, Integrated DNA Technologies, Cat# 1075916), at a density of 1 x 10^6 cells/20µL. NK cells were transferred into Lonza electroporation 16-well Nucleocuvettes (P3 Primary Cell 4D-Nucleofector™ X Kit S, Lonza Bioscience, Cat# V4XP-3032) and electroporated using pulse code CM-137. Post electroporation, NK cells were recovered with 100 µL prewarmed NK cell medium and rested for 10-15 minutes in the incubator at 37°C. NK cells were then transferred to cell culture flasks containing irradiated uAPC cells at an E:T ratio of 1:2, suspended in prewarmed NK cell medium, supplemented with IL-2 (400U/mL). 

Transcription factor library CRISPR screens in primary human NK cells
Primary human NK cells were retrovirally transduced with a transfer plasmid containing the transcription factor library (11,364 unique sgRNAs) on day 5 post isolation from cord blood. NK cells which had stably integrated a copy of the vector plasmid following low-MOI (0.3) transduction were subsequently selected using puromycin at a concentration of 2.5µg/mL. On day 11, library-transduced NK cells were electroporated with recombinant Cas9 protein (18.3µM, Alt-R™ S.p. Cas9 Nuclease V3, Cat# 1081059) in the presence of P3 primary buffer using the Lonza 4D Nucleofector (P3 Primary Cell 4D-Nucleofector X Kit L, Cat # V4XP-3012). Immediately after electroporation, NK cells were recovered using prewarmed NK cell medium, rested in the incubator for 10-15 min at 37°C and subsequently re-expanded with uAPCs at an E:T ratio of 1:1. T0 samples were collected on day 15.

Pooled genome-wide CRISPR screening in primary human NK cells

On day 5 post isolation, primary human NK cells from each donor were transduced with the retroviral vector expressing the genome-wide sgRNA library at low MOI (0.3) at a coverage = 500x in RetroNectin-coated plates (Takara, Cat# T100B). Starting day 7, NK cells stably expressing the CRISPR library vector, were selected using puromycin (Gibco; Cat# A1113803) at a concentration of 2.5µg/mL for a duration of 5 days, replenishing puromycin every 48h. Puromycin-selected NK cells were transfected with
Cas9 by electroporation using the Lonza 4D nucleofector device. In brief, NK cells were washed with PBS, pelleted, and resuspended in P3 primary buffer, supplemented with Cas9 (10.16µM; Alt-R™ S.p. Cas9 Nuclease V3, Integrated DNA Technologies, Cat# 10000735) and electroporation enhancer (4.16µM; Alt-R™ Cas9 Electroporation Enhancer, Integrated DNA Technologies, Cat# 1075916), at a density of 1 x 10^7 cells/100 µL. NK cells were transferred into Lonza electroporation Nucleocuvettes (P3 Primary Cell 4D-Nucleofector™ X Kit L, Lonza Bioscience, Cat# V4XP-3024) and electroporated using pulse code CM137. After electroporation, NK cells were recovered with 500 µL prewarmed NK cell medium and rested for 10-15 minutes in the incubator at 37°C. Subsequently, NK cells transferred to cell culture flasks containing irradiated uAPC cells suspended in prewarmed NK cell medium, supplemented with IL-2 (400U/mL).