Cas9 h840aprotein

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).