Fastdigest bpil

Antibodies to RyR (pan-specific), AHCY, AHCYL1, and β-actin, and mouse IgG-κ binding protein conjugated to CFL 488 were from Santa Cruz Biotechnology (Dallas, TX). Goat anti-mouse IgG conjugated to IRDye 680RD and goat anti-rabbit IRDye 800CW were from LI-COR (Lincoln, NE). Antibodies to phosphatidylinositol 4,5 bisphosphate (PIP₂) were from Echelon Biosciences (Salt Lake City, UT). Goat anti-mouse IgG conjugated to horseradish peroxidase was from BioRad (Hercules, CA). ERK1/2 and pERK1/2 antibodies were from Cell Signaling Technology (Danvers, MA). Oligonucleotides encoding gRNA and primers were obtained from Integrated DNA Technologies (Coralville, IA). The pSpCas9(BB)-2A-Puro (PX459) V2.0 vector was a gift from Feng Zhang (Addgene plasmid # 62988). T4 polynucleotide kinase and T7 DNA ligase were from New England Biolabs (Ipswich, MA). Fast AP thermosensitive alkaline phosphatase and FastDigest Bpil were from Thermo Scientific (Waltham, MA). Plasmid-Safe ATP-Dependent DNase was from Lucigen (Middleton, WI). Surveyor Mutation Detection Kit S100 was from Integrated DNA Technologies (Coralville, IA).
Chemicals- Fura-2 AM was from Invitrogen (Carlsbad, CA). Xestospongin C was from Cayman Chemical (Ann Arbor, MI). All other reagents, unless otherwise indicated, were from Sigma-Aldrich (St. Louis, MO).

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.