Mammalian Cell Expression Plasmid Construction

All mammalian cell expression plasmids were constructed by USER cloning from gBlock gene fragments (Integrated DNA Technologies) with USER junctions sized between 14 and 20 nucleotides^{58 (link)}. Phusion U Green Multiplex PCR Master Mix (ThermoFisher) was used for amplification of DNA. sgRNA plasmids were constructed by blunt end ligation of a linear PCR product generated by encoding the 20-nt variable protospacer sequence onto the 5′ end of an amplification primer and treating the resulting piece to KLD Enzyme Mix (New England Biolabs) according to the manufacturers’ instruction. Mach1 chemically competent E. coli (ThermoFisher) cells were used.