CRISPR/Cas9 editing
DNAM-1 in parental 221 and CD155-transfected cell lines,  as well as CD155 and CD112 from K562 and 293T cells, was  edited using CRISPR/Cas9 technology. In brief, ribonucleoprotein (RNP) complexes were formed between synthetic guide  RNA (sgRNA) (CD226þ69947029 and CD226- 69947061;  PVRþ44647235 and PVR44647223; NECTIN2- 44865272  and NECTIN2- 44865280 from Synthego) and Cas9 protein  (Integrated DNA Technologies) and then added to 2 × 105 cells resuspended in Nucleofector Solution SF and Supplement  1 (Lonza) before nucleofection with the 4D-Nucleofector X  Unit, program CM130. Cells were sorted where necessary to 
achieve DNAM-1 or CD155/CD112 null cell lines. For editing  of primary NK cells, 1 × 106 NK cells resuspended in P3 Nucleofector media and Supplement 1 (P3 Primary Cell 4DNucleofector X Kit S, Lonza) were incubated with Cas9 protein and sgRNA before nucleofection with the 4D Nucleofector X Unit, program CM137. The sgRNAs used  for NK cells were DNAM-1: CD226þ69947029  and CD226- 69947061; TIGIT: TIGITþ114295552 and  TIGIT114295545; CD96: CD96þ111545092 and  CD96- 111545121; and NKG2D: KLRK1- 10386955 and  KLRK1- 10386987. NK cells were allowed to recover for 4  days at 37 °C, 5% CO2 with 100 U/mL IL-2 before sorting for  receptor negative populations, staining for CD56, CD3,  DNAM-1, TIGIT, NKG2D, and/or CD96, along with Fixable  Viability Dye eFluor 780. Sorted NK cells were then plated  onto irradiated PBMCs and 221 cells transfected with HLA-G,  along with 100 U/mL IL-2 and 1.5 ng/mL phytohemagglutinin  (Gibco Ltd) as described previously.34 Cells were expanded for  2 to 3 weeks before use in functional NK cell assays.