Electroporation and dual luciferase reporter assay
gBlocks with selected SNP-containing fragments (IDT) were cloned into pGL4.10 plasmid (Promega). For the luciferase reporter assay, 150 K Caco2, Jurkat cellswere nucleofectedwith 50 ng of Renilla vector along with 170 ng of luciferase vectors using the Lonza Nucleofector according to the manufacturer’s protocol.

Primary macrophage CRISPR-Cas9 mediated genome editing
CD14+ monocyte cells were thawed and plated in growth media. On day 5, adherent cells were collected over ice using a cell lifter. The cells were washed once with Phosphate-buffered saline (PBS) and resuspended in P3 Primary Cell solution (Lonza). RNP complexes were formed by mixing Cas9 protein (IDT, 1081058) with the indicated sgRNA and incubating at room temperature for 10 min. RNP complexes were then added to the adherent cell solution and electroporated using the Lonza 4D-Nucleofector and protocol CA-137. The cells were then transferred into media and incubated at 37 °C, 5% CO2. After 3 days, cells were treated with LPS for times indicated.

Nucleofection of Cas9/gRNA RNP complex and HDR repair template
RNP complex containing gRNA and Cas9 protein (IDT, Alt-R® S.p. Cas9 Nuclease V3, Cat #: 1081058) were prepared by mixing 150 pmol gRNA and 125 pmol of the Cas9 enzyme in 1.5 µL PBS to a total 5 µL reaction volume per well of a 96-well plate. The reaction was incubated at room temperature for 20 min. U937 cells (100,000 cells/well) were washed twice with PBS and resuspended in a solution containing a 4.5:5 mixture of nucleofection buffer (Buffer SE, Lonza Lot #: S-09279) and supplement (Lonza, Lot #: 09588). The Cell suspension was mixed with the 5 µL of RNP complex, 100 µM HDR template, 100µM Alt-Cas9 electroporation enhancer (IDT cat# 10007805), and PBS to a final volume of 30 µL. The nucleofection mixture was transferred to a 96- well Nucleocuvette (25 µL/well) and electroporated using Lonza 96- well Shuttle following the FP-100 program. After electroporation, the cells were transferred to a 96-well tissue culture plate containing 1.0 µM Alt-R HDR Enhancer V2 (IDT, Cat #: 10007921) in 200 µL of cell culture media.