Neb 10 β competent cells

All BE plasmids were constructed with USER cloning (Badran et al., 2016 (link)) with pCMV ABEmax_P2A_GFP (Addgene #112101) and pCMV_AncBE4max_P2A_GFP (Addgene #112100) plasmids as template, using Phusion U Hot Start Polymerase (ThermoFisher Scientific). All sgRNA expression plasmids were generated using blunt-end cloning with pFYF1230 (Addgene plasmid #47511) as a template, using Phusion High-Fidelity DNA Polymerase (New England BioLabs). All DNA vector amplification was carried out using NEB 10-β competent cells (New England BioLabs). All plasmids were purified using the ZymoPURE II Plasmid Midiprep Kit (Zymo Research D4200).

Human SOD1 cDNA’s with mutations described in this study were generated using oligonucleotide primers encoding the desired mutation with Quick-Change mutagenesis kits. The single mutants were made using pEF.BOS vectors [68 (link)] that encode WT human SOD1 (WT-hSOD1) as the template [49 (link)]. The protocol used to make mutations used a modified PCR strategy with primers encoding specific mutations and pEF-BOS-WT SOD1 or pEF-BOS-WT SOD1:YFP plasmids as the template. The PCR reaction used Platinum Pfx polymerase (Invitrogen/ThermoFisher, Waltham, MA) and 2X Pfx buffer concentration to accommodate the large plasmids that were amplified. The PCR reaction products were digested with Dpn1 to remove template and then transformed into NEB-10β competent cells (New England Biolabs, Ipswich, MA) following standard protocols. Large scale preparations of plasmid DNA for transfection were prepared by CsCl gradient purification. The SOD1 and SOD1:YFP coding sequence of all plasmids was verified by DNA sequence analysis.