Phusion u dna polymerase green multiplex pcr master mix

Constructs used in this study were obtained by USER assembly, Gibson assembly, or synthesized by Genscript. Gene fragments used for PCR were purchased as mammalian codon-optimized gene fragments from IDT, Thermo Fisher Scientific and Twist Bioscience. PCR were performed with primers ordered from IDT using either Phusion U DNA Polymerase Green MultiPlex PCR Master Mix (Thermo Fisher) or Q5 Hot Start HiFi 2x Master Mix (New England Biolabs). Endo-free plasmids used for mammalian transfection were prepped using ZymoPURE II Plasmid Midiprep (Zymo Research Corporation) from 50 mL Mach1 (Thermo Fisher) culture. mRNA was synthesized by IVT reactions, and gRNA with standard modification was purchased from Axolabs. Sequences for CBEs, protospacer sequences for sgRNA, and oligos used in this study can be found in Supplementary Table 1–4. Prism8 (v 8.3.0), Excel (v16.32), R (v3.4.3) were used for data analysis in addition to data analysis listed in Supplementary Note 1 and 3–5.

All molecular biology methods and cloning steps were performed as previously described^{13 (link)}, including the utilization of USER enzyme (New England Biolabs, NEB, M5505L), Phusion U DNA Polymerase Green Multiplex PCR Master Mix (Thermo Fisher Scientific, F564L), Q5 Hot Start High-Fidelity 2X Master Mix (NEB, M0494L), Mach T1 competent cells (Thermo Fisher Scientific, C8681201) and ZymoPURE II Plasmid Midiprep kits (Zymo Research Corporation, D4201) in accordance with manufacturers’ protocols. Amino acid sequences for base editors highlighted in this study can be found in Supplementary Sequences 3–31. Sequences of sgRNAs used to target genomic sites can be found in Supplementary Table 3. Representative CABE-Ts and CBE-Ts used in this study have been deposited on Addgene.