CRISPR-Cas9 Genome Editing Protocol

BACs were purchased from Thermo Fisher Scientific (Waltham, MA) in the case of BAC CTD-2166E9, and from the BACPAC Resources Center (Children’s Hospital Oakland Research Institute, CA) in the case of all other BACs. BACs were purified from E. coli using the Nucleobond Xtra BAC Maxi Kit (Macherey-Nagel, Duren, Germany). SP-dCAS9-VPR (Addgene ID: 63798) was provided by the Qi lab [19 (link)], and the sgRNA-encoding plasmid along with the Cas9 plasmid, pMCB306 (Addgene ID: 89360) and lentiCas9-Blast (Addgene ID: 52962) respectively, were gifts from Michael Bassik [30 (link),31 (link)]. Guides were cloned into the pMCB306 BlpI/BstXI sites using annealed oligos with the appropriate sticky ends (S1 File).