Phusion u green multiplex pcr master mix

PCR was performed using Phusion U Green Multiplex PCR Master Mix (ThermoFisher Scientific). All plasmids were assembled by the USER cloning method as previously described^{2 (link)}. The amino acid sequences for maxed base editor variants are listed in Supplementary Sequences 1. Guide RNA plasmids for SpCas9, SaCas9, and all engineered variants were assembled as previously described^{10 (link)}. Sequences of protospacers used in this study are listed in Supplementary Table 8. Plasmids for mammalian cell transfections were prepared using the ZymoPURE Plasmid Midiprep kit (Zymo Research Corporation).

DNA amplification was conducted by PCR using Phusion U Green Multiplex
PCR Master Mix (ThermoFisher Scientific) or Q5 Hot Start High-Fidelity 2x Master
Mix (New England BioLabs) unless otherwise noted. DNA oligonucleotides,
including Cy5-labeled DNA oligonucleotides, dCas9 protein, and Cas9 H840A
protein were obtained from Integrated DNA Technologies. Yeast reporter plasmids
were derived from previously described plasmids³⁹ and cloned by the Gibson assembly
method. All mammalian editor plasmids used in this work were assembled using the
USER cloning method as previously described⁴⁰ . Plasmids expressing sgRNAs were constructed by
ligation of annealed oligonucleotides into BsmBI-digested
acceptor vector (Addgene plasmid #65777). Plasmids expressing pegRNAs were
constructed by Gibson assembly or Golden Gate assembly using a custom acceptor
plasmid (see Supplementary
Note 3). Sequences of sgRNA and pegRNA constructs used in this work
are listed in Supplementary
Tables 2 and 3. All vectors for mammalian cell experiments were purified using
Plasmid Plus Midiprep kits (Qiagen) or PureYield plasmid miniprep kits
(Promega), which include endotoxin removal steps. All experiments using live
animals were approved by the Broad Institute Institutional and Animal Care and
Use Committees. Wild-type C57BL/6 mice were obtained from Charles River
(#027).

PCR was performed using either Phusion U Green Multiplex PCR Master Mix (ThermoFisher Scientific) or Q5 Host Start High-Fidelity 2x Master Mix (New England Biolabs) unless otherwise noted. All plasmids were assembled by either the USER cloning method as previously described^{20 (link)} or by Gibson assembly^{21 (link)}. Plasmids for mammalian cell transfections were prepared using an endotoxin removal plasmid purification system, ZymoPURE Plasmid Midiprep (Zymo Research Corporation).