Validation of individual knockout efficiency

We validated the knockout efficiency for seven individual genes in qNSC cultures in two independent experiments:

For experiment 1, young and old NSCs were seeded in a 24-well PDL pre-coated plate at a density of 2–3 × 10⁵ cells per well and incubated in qNSC medium. After 4 days with medium changes every 2 days, qNSCs were infected with lentiviruses expressing sgRNAs targeting each gene (5 sgRNAs per gene) as described in the section ‘Assessment of the impact of individual gene knockout on NSC activation’ (see Source Data Extended Data Fig. 1n for sgRNA sequences). Six days after infection, cells were washed with PBS then lysed directly with DirectPCR Lysis reagent (Viagen Biotech, 102-T) with 1% Proteinase K (Fisher Scientific, 25-530-049) for 10 min at room temperature. The supernatant was pipetted repeatedly, then transferred to PCR strip tubes and incubated at 65 °C for 25 min, and then 95 °C for 15 min in a thermocycler. We amplified genomic DNA with primer pairs surrounding the sgRNA-editing sites (see Source Data Extended Data Fig. 1n for amplification primers), using Q5 polymerase (Fisher Scientific, M0491L) and the following program: 30 s of annealing step at 55 °C and 1 min of extending step at 72 °C for 40 cycles total.

For experiment 2, we cloned 5 sgRNAs for each individual gene, using the same methodology as described in the section ‘sgRNA plasmid sublibrary cloning for in vivo screens’ above. For lentiviral production, 293T cells were seeded in DMEM + 10% FBS (Gibco 10099141) + 1× penicillin–streptomycin–glutamine (Gibco, 10378-016) at a density of 13 × 10⁶ cells in 15 cm plates. One day later, 293T medium was replaced with 18 ml fresh medium and the cells were transfected using the PEI (1 mg ml^–1, Polysciences, 23966-2) transfection method. The individual gene library (25.5 µg) was transfected together with the lentiviral packaging plasmids psPAX2 (32.12 µg) and pCMV-VSV-G (9.44 µg) per 15 cm plate. psPAX2 was a gift from D. Trono (Addgene, plasmid 12260; http://n2t.net/addgene:12260; RRID:Addgene_12260). pCMV-VSV-G was a gift from B. Weinberg (Addgene, plasmid 8454; http://n2t.net/addgene:8454; RRID:Addgene_8454). One day (20–24 h) after transfection, the medium was changed to Neurobasal A with penicillin–streptomycin–glutamine. After another 20–24 h, lentivirus containing supernatant was collected and stored at 4 °C and fresh medium was added to the 293T cells for another collection after 24 h. Both supernatants were then combined, filtered through a 0.45 µm polyvinylidene fluoride filter (Millipore Sigma, SE1M003M00) and frozen at −80 °C in 5 ml aliquots. For lentiviral transduction, young qNSCs were plated onto 6-well PDL pre-coated plates at a density of 1.75 × 10⁶ cells per well (for control lentivirus), 10 cm PDL pre-coated plates at the density of 1.0 × 10⁷ cells per plate (for Slc2a4-targeting lentivirus) or 12-well PDL pre-coated plates at a density of 4.0 × 10⁵ cells (for Npb and B3galnt2 targeting lentivirus). NSCs were kept in qNSC medium for 4 days (with medium changes every other day) before transduction. After removing medium, viral supernatants (2 ml for 6-well plates, 10 ml for 10 cm plates and 1 ml for 12-well plates) were thawed at room temperature and mixed with 8% of B27 minus vitamin A, bFGF (80 ng ml^–1) and BMP4 (200 ng ml^–1). qNSCs were incubated with lentiviral medium for 24 h. After removing lentiviral medium after 24 h, a second lentiviral transduction was repeated the next day. After two consecutive transductions, qNSCs were washed once with Neurobasal A medium and then cells were kept in qNSC medium for 7 days to allow recovery and CRISPR editing. To select for a population of cells that was infected by the lentivirus, 1.0 μg ml^–1 of puromycin (Sigma-Aldrich, P8833) was added to the cultures for 3 days, with medium changes every day. To assess knockout efficiency, we isolated genomic DNA as described above for experiment 1. We amplified genomic DNA with primer pairs roughly 150–250 bp upstream and 300–450 bp downstream of sgRNA editing site (see source data for list of primers) using GoTaq Green master mix (Promega, M7123) and the following amplification program: 30 s of annealing step at 55 °C and 1 min of extending step at 72 °C for 40 cycles total.

In both experiment 1 and experiment 2, PCR amplicons were Sanger sequenced using the respective forward primers (source data). We then analysed knockout efficiency using the DECODR (v.3.0) online tool (https://decodr.org/)⁹³. Each sgRNA was analysed separately, and the editing efficiency is indicated in source data. Individual sgRNAs that had an editing efficiency with a r² value less than 0.6 from DECODR (v.3.0) are indicated as low confidence (LC) in source data and marked with a hash symbol in Extended Data Fig. 1n. Individual sgRNAs that were not detected by DECODR (v.3.0) in the Sanger sequencing trace are indicated as not detected (ND) in source data and not included as data points in Extended Data Fig. 1n. Finally, we note that the percentage of knockout per gene is probably also underestimated due to the fact that larger indels that span sgRNA cutting sites are not taken into account by DECODR.