Crisprmax cas9 transfection reagent

U87MG/CPOX-KO cells were created by transfection of ribonucleoprotein complexes including Cas9 and a mixture of three single-guide RNAs (sgRNAs)^{65 (link)} targeting an early exon of the CPOX gene (Synthego). U87MG cells were seeded at a density of 2 × 10⁵ cells per well (six-well plate). After 24 h incubation, the cells were transfected with a mixture of sgRNAs, Cas9 and the CRISPRMAX-Cas9 transfection reagent (Cat# CMAX00008, Thermo Fisher Scientific) in serum-free OptiMEM (Cat# 31985070, Thermo Fisher Scientific). After 48 h, media containing the transfection reagent was replaced with fresh media (Media #2, Supplementary Table 1) and allowed to grow for another 2 days. Validation of gene targeting (knockout, KO) was then confirmed by immunoblot using whole-cell lysates, as compared to a non-targeted control. CPOX antibody (Novus Biologicals, cat# NBP2-59438) was used to confirm the loss of CPOX protein expression after KO and α-Actinin antibody (Cell Signaling, cat# 12413S) was used to confirm expression of the immunoblot loading controls.

LN428/ORC2-KO cells were created by transfection of ribonucleoprotein complexes including Cas9 and a mixture of three single-guide RNAs (sgRNAs) (41 (link)) targeting an early exon of the ORC2 gene (Synthego). LN428 cells were seeded at a density of 2 × 10⁵ cells per well (six-well plate). After 24 h incubation, the cells were transfected with a mixture of sgRNAs, Cas9 and the CRISPRMAX-Cas9 transfection reagent (Cat# CMAX00008, Thermo Fisher Scientific) in serum-free OptiMEM (Cat# 31985070, Thermo Fisher Scientific). After 48 h, media containing the transfection reagent were replaced with fresh media (Media #2, Supplementary Table S1) and allowed to grow for another 2 days. Validation of gene targeting (knockout, KO) was then confirmed by immunoblot using whole cell lysates, as compared to a non-targeted control. The primary and secondary antibodies used are listed in Supplementary Table S2.

Primary human fibroblasts stably expressing mGFP or IFIX-mGFP were generated previously in our laboratory via retrovirus transduction with pLXSN plasmids (24 (link)). The inducible IFIX-GFP 293 cells were generated previously in our laboratory by cotransfection with pcDNA5/FTR/TO plasmids (11 (link)). IFIX-KO and control HFF cell lines were produced using TrueCut Cas9 protein V2 (Thermo Fisher Scientific) and CRISPRMAX Cas9 transfection reagent (Thermo Fisher Scientific) with TrueGuide synthetic guide RNA (negative control, nontargeting 1; PYHIN1, CRISPR728173_SGM; Thermo Fisher Scientific) according to the manufacturer’s instructions.