Generating Knockout Monocytes using CRISPR-Cas9

Nontargeting,
SLC46A2, and SLC46A3 sgRNAs were purchased from IDT and resuspended
to 100 μM in TE buffer. Cas9 RNPs were formed by adding 8 μL
of 61 μM Alt-R S.p. Cas9 Nuclease V3 (IDT) to 12 μL of
100 μM sgRNA and incubating for 10 min at room temperature.
Freshly purified CD14⁺ monocytes were washed once with
cold PBS, then resuspended in P3 Primary Cell nucleofector solution
(Lonza) to a density of 10⁷ cells/100 μL. A volume
of 100 μL of resuspended monocytes was then added to the Cas9
RNPs, transferred to a nucleofection cuvette, and nucleofected using
program CM-137 on a Nucleofector 4D device (Lonza). Electroporated
cells were then transferred to a six-well plate containing 2 mL of
DMEM with 10% heat-inactivated FBS and 1% penicillin-streptomycin.
At 24 h after nucleofection, cells were pelleted and resuspended in
2 mL of fresh media. At 72 h after transfection, cells were used for
CDN stimulation assays and genomic DNA was isolated to measure the
knockout efficiency. The knockout efficiency was determined by amplifying
the region of genomic DNA surrounding sgRNA target sites (using the
primers listed in Table S1), performing
Sanger sequencing, and using the sequencing trace to estimate knockout
efficiency through TIDE analysis.^{69 (link)}