Pmcb306

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).

SkHep1 and A549 cell lines stably expressing Cas9 endonuclease (SkHep1-Cas9⁺ and A549-Cas9⁺) are gifts from Junwei Shi (University of Pennsylvania). MCF7 cell line stably expressing Cas9 (MCF7-Cas9⁺) was purchased from Applied Biological Materials (Cat # T3257). Both SkHep1-Cas9⁺ and A549-Cas9⁺ cells were maintained in DMEM media and MCF7-Cas9⁺ cells were maintained in PriGrowIII media (TM003, abm). All cell culture media was supplied with 10% FBS, 100 unit/ml penicillin and 100 μg/ml streptomycin.
sgRNAs targeting test genes were designed using the DESKGEN software (https://www.deskgen.com/landing/), and sequences for non-targeting control sgRNAs were chosen from published literature^{59 (link)}. The sgRNAs targeting test genes or control sgRNAs were individually cloned into a lentiviral vector, either pMCB306 (Addgene: #89360) which expresses the GFP reporter gene or pMCB320 (Addgene: #89359) which expresses the mCherry reporter gene. All cloned sgRNA constructs were verified by Sanger sequencing.