Ampicillin

BamHI-HF, XhoI, T4 DNA ligase and DNA polymerase were obtained from New England Biolabs (USA). Unstained Protein Ladder, Monarch ® Plasmid Miniprep Kit and Monarch ® DNA Gel Extraction Kit were also purchased from New England Biolabs (USA). Agar, peptone, yeast extract, sodium chloride, ampicillin and IPTG were obtained from Bioline (UK). All chemicals used in SDS-PAGE were purchased from Merck (Germany). Fructosyl valine (FV) was prepared as previously described (Keil, Mortensen, & Christophersen, 1985; (link)Rajkumar, Warsinke, Möhwald, Scheller, & Katterle, 2007) (link). Sodium hydroxide, sodium acetate, valine, and glucose were purchased from Sigma-Aldrich.

Single guide RNA (sgRNA) sequences for non-targeting control and ACE2 were taken from the Weissman Human Genome-wide CRISPRa-v2 library (Addgene #83978). LRRC15 sgRNA sequences and additional ACE2 sgRNA sequences were taken from the Human CRISPR activation pooled library set A (Addgene #92379). Sense and antisense strands for each sequence were ordered as DNA oligonucleotides (IDT) with 5' overhangs of 5'-CACC-3' on the sense strand oligonucleotide and 5'-AAAC-3' on the antisense strand oligonucleotide. Oligonucleotides were annealed at 4°C for 16 h and pXPR-502 (Addgene #96923) was digested with Esp3I (ThermoFisher Scientific, ER0451) or BsmBI-v2 (New England Biolabs). sgRNA DNA oligonucleotide duplexes were ligated into the digested pXPR-502 backbone using T4 ligase (New England Biolabs) and incubated at 4°C overnight. NEB 10-beta competent E. coli (New England Biolabs) were transformed with 100 ng of each sgRNA construct by heat-shock, plated onto LBagar plates (Life Technologies) containing ampicillin (Sigma-Aldrich) and grown at 37°C. Individual colonies were picked, expanded in Luria broth (Life Technologies) supplemented with ampicillin and amplified constructs were harvested using either ISOLATE II Plasmid Mini Kit (Bioline) or PureYield Plasmid Maxiprep System (Promega Corporation).