Ribonucleoproteins (RNPs) were preassembled at a 1:3 molar ratio by incubating 18.3 pmol (3 mg) of Cas9 protein (IDT, Coralville, Iowa) with 55 pmol of gRNA (Biolegio, Nijmegen, The Netherlands) (Table E1 in this article’s Online Repository at www.jacionline.org) for 10 minutes at room temperature. Nucleofections were performed with RNPs or 1 mg of PureBoost EGFPmRNA (Cellerna, Baesweiler, Germany) using a 4D-Nucleofector (Lonza, Basel, Switzerland).
Functional T-cell assays
For in vivo evaluation, CD81 T cells were purified from splenocytes (StemCell Technologies, 19853, Vancouver, Canada), activated for 3 days with beads coated with CD3e and CD28 antibodies (Miltenyi Biotec, 130-093-627, Bergisch Gladbach, Germany) in RPMI 1640 medium (Gibco, Life Technologies, Waltham, Mass) supplemented with 10% FCS (PAA, BioPath Stores, Cambridge, UK), Pen/Strep (Sigma-Aldrich, St Louis, Mo), and murine IL-2 (10 U/mL) (Peprotech, Hamburg, Germany) before nucleofection (in P4 with CM-137).
Transplantation of gene-edited HSCs and LCMV challenge
Lineage-negative HSCs (Miltenyi Biotec, 130-090-858. Bergisch Gladbach, Germany) from femur and tibia-derived bone marrow (BM) were stimulated for 3 hours in StemSpan SFEM II medium (StemCell Technologies, Vancouver, Canada) supplemented with L-glutamine (20 mM) (Gibco, Life Technologies), mSCF, mTPO, and hIGF2 (20 ng/mL each) plus hIGF2 and mIL-3 (10 ng/mL each) (Peprotech. Hamburg, Germany) before nucleofection (in P4 with CA-137).
Jinx reporter cell line
K562 cells (ATCC) were used to create a Jinx reporter cell line using a targeted integration strategy. E1 Briefly, the Unc13d intron 26 sequence present in Jinx mice was inserted into position 146 of plasmid pEGFP-C1 (Clontech) via Gibson assembly E2 to generate pEGFP-intron26-Unc13d that harbors homologous sequences to the AAVS1 region. Integration into AAVS1 was achieved by CRISPR-mediated knock-in (4D-Nucleofector, Lonza, SF FF-120).