E coli gt116 cells

Guide RNA sequences for the CRISPR/Cas9 system were designed as described in Ran et al. with the use of CRISPOR software (http://crispor.tefor.net/crispor.py; Haeussler et al., 2016 (link)). Briefly, the top and bottom strand of 20-nt guide RNA were synthesized (IBB, Warsaw), annealed and ligated into the pair of FastDigest BsmBI (Thermo Fisher Scientific, Waltham, MA, USA) cut plasmids, namely, pSpCas9(BB)-2A-GFP (PX458) (Addgene, Cambridge, MA, USA) and its nickase version (D10A nickase mutant; pSpCas9n(BB)-2A-GFP (PX461)) from S. pyogenes (Ran et al., 2013 (link)). 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 Scientific) and verified with Sanger sequencing. For larger scale plasmid preparations, the Qiagen Midi kit was used (Qiagen, Hilden, Germany). The sgRNA oligonucleotide sequences are presented in Table S1.

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.