The guide RNAs specific for the HTT gene (HTT_sg1, HTT_sg3 and HTT_sg4) were previously described and validated [26 (link),32 (link),35 (link)]. The top and bottom strands of the 20-nt guide RNAs (Table S5 ) were synthesized (IBB, Warsaw, Poland), annealed and ligated into the pair of FastDigest Bpil (Thermo Fisher Scientific) cut plasmids: pSpCas9(BB)-2A-GFP (PX458) and its nickase version (D10A nickase mutant; pSpCas9n(BB)-2A-GFP (PX461)) (Addgene, Cambridge, MA, USA) from S. pyogenes [36 (link)]. The ligated products were transformed into chemically competent E. coli GT116 cells (InvivoGen, San Diego, CA, USA), and the cells were plated onto ampicillin selection plates (100 μg/mL ampicillin) and incubated at 37°C overnight. Plasmid DNA was isolated using the Gene JET Plasmid Miniprep kit (Thermo Fisher Scientific) and verified with Sanger sequencing. Electroporation was used to deliver Cas9 protein, HTT_sgRNA and a donor template for HDR.
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