Lenticrispr v2 vector system

CRISPR/Cas9 gene editing was performed using the LentiCRISPR v2 vector system (Addgene, Plasmid #52961) with the gRNA sequences listed in Supplementary Table S1. Lentiviruses were infected into human Hela cells. Single puromycin-resistant clones were selected using limited-dilution cloning in 96-well plates. Genomic DNA was isolated from each clone and DNA fragments spanning S1606 and S961 were amplified and sequenced. Cells positive for mutations, except for the homozygous mutations, were subcloned and the same DNA fragments were cloned into TA vectors for sequencing of both alleles.

CRISPR–Cas9 was performed using the LentiCRISPR v.2 vector system (Addgene). Lentiviruses were infected into the human microglia cell line HMC3. Single, puromycin-resistant clones were selected using limited-dilution cloning in 96-well plates. Genomic DNA was isolated from each clone, and DNA fragments crossing fSNP rs1537371 were amplified and sequenced. Cells positive for mutations, except for homozygous mutations, were subcloned and the same DNA fragments were cloned into pGEM-T Easy vector (Promega) for sequencing of both alleles. For control, we used polyclonal cells targeted by the same CRISPR–cas9 targeting vector containing a guide RNA sequence irrelevant to rs1537371.

To generate guide RNA (gRNA) targeting GCN2 expression, we employed the LentiCRISPR v.2 vector system (Addgene, Watertown, MA, USA). Constructs were designed to maximize on-site specificity and minimize off-target activity using a publicly-available online CRISPR design algorithms^{72 (link)}. RNA guides were cloned into LentiCRISPR v.2 puromycin-resistant backbone and selected by ampicillin resistance in Stbl3 bacteria according to the Lentiviral CRISPR Toolbox standard protocol from the Zhang Lab^{73 (link),74 (link)}. GCN2 guide and control RNA lentiviral particles were produced by transfecting HEK293T cells (obtained from ATCC) using the Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, Burlington, ON, Canada). The medium was replaced with IMDM medium containing 10% FBS 12 h after transfection, and medium containing viral particles was collected 48 and 72 h after initial transfection. Media was filtered using a 0.45 μM filter, aliquoted, and stored at −80 °C. DNA oligonucleotides used for GCN2 knockout experiments are described in Supplementary Table 1.