4.6. CRISPR/Cas9 Editing Genome SOD1 and YWHAZ in HEK293T Cells

HEK293T cells (10⁶ seeded on T-25 culture flask) were transiently transfected using lipo3000 with the pX459 vector (gifted by Dr. John Schimenti, Cornell University, Ithaca, NY, USA) integrated with the guide RNA (gRNA) targeting 5′- CTAGCGAGTTATGGCGACGA-3′ sequence in exon 1 of the SOD1 gene (as pX459-SOD1), or pDG459 (gifted by Dr. Tudorita Tumbar, Cornell University, Ithaca, NY, USA) integrated the dual gRNAs targeting 5′-CATGACTGGATGTTCTGCAG-3′ and 5′-AGATATCTGCAATGATGTAC-3′ sequences in exon 4 of the YWHAZ gene (as pDG459-YWAHZ) following standard cloning method described in the protocol [73,74] to generate stable SOD1 knockdown (SOD1-KD) or YWHAZ knockout (YWHAZ-KO) cells. The SOD1-A4V (alanine to valine at fourth position) genome mutation was created by homologous recombination repairing of CRISPR-Cas knock-in through adding single-stranded oligodeoxynucleotides (ssODNs) as a donor template during the pX459-SOD1 transfection period. We only screened out SOD1-KD cells because SOD1 knockout seemed lethal to our HEK293T cells. We constructed the SOD1-A4V mutant in the cell line because the A4V mutant is also related to ALS, which depleted SOD1 activity by 97% (Figure S4D) and is the most common mutant with rapid disease progression in US ALS patients [75]. In this way, we could generate both SOD1-KD and SOD1-A4V as protein-loss and ALS-related cell lines to test intracellular conditions under impaired SOD1 protein and activity.

Primer sets used for the annealing formation of 20 bp gRNA oligonucleotides were listed in Table S2. At 48 h after transfection, cells were selected using puromycin (0.8 μg/mL) for another 72 h. The cells were harvested, washed 3 times with PBS, and resuspended in FACS buffer (1L PBS, 1g BSA, 2mM EDTA) to a cell density of 10⁶. The cells were run through SONY MA900 flow cytometry (Core Facility, Cornell University, Ithaca, NY, USA) and sorted out as single cells into the 96-well plate. After the single colony grew, part was used to extract DNA by an alkaline lysis method [76], followed by colony PCR (primers in Table S2) and sequencing of exon 1 of the SOD1 gene or exon 4 of the YWHAZ gene. The rest of the colony was maintained under standard culture and passage. The heterozygotes could continue to perform the TA cloning (TOPO™ TA Cloning™ kit, Thermo Fisher Scientific, Waltham, MA, USA) on colony PCR products to confirm the sequence information for each chromosome. Successfully edited colonies were maintained for subsequent experiments. The SOD1 protein knockdown cells were further transfected with the pDG459-YWHAZ vector and followed the same process to select SOD1 protein knockdown and YWHAZ protein knockout cells.