Generation of Cas9-Cre Bicistronic Plasmid

The pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (a gift from Feng Zhang; Addgene plasmid #42230) was modified to express a bicistronic peptide containing Cas9 and Cre as follows. pX330 was sequentially modified to accept a 2.4 kb Cas9-P2A-Cre fragment from pSECC (a gift from Tyler Jacks; Addgene plasmid #60820) first by an EcoRI (New England Biolabs) digestion with a subsequent fill-in reaction by DNA polymerase and then a AccIII restriction digest. The 2.4 kb insertion fragment was generated from pSECC by a restriction digest with AccIII (Promega) and SacII (New England Biolabs). Ligation of this fragment into the pX330 modified plasmid was performed using T4 DNA Ligase (Invitrogen) in an overnight reaction at 16°C. The ligated product was then used to transform competent bacteria. sgRNAs targeting Trim24, Kif5b, Tpm3, Ret and Ntrk1 were designed using the Zhang lab CRISPR design tool (crispr.mit.edu) and are presented in Table S1. The pX330+Cre plasmid was digested with BbsI (New England BioLabs) and ligated to annealed and phosphorylated sgRNA oligonucleotides (Integrated DNA Technologies). Ligated plasmids were transformed into DH5α E.coli (Life Technologies). The PX330-Alk-Eml4 plasmid was kindly provided by Andrea Ventura (Maddalo et al., 2014 (link)).