Pcmv ha3a be3 y130f

The mRNA transcriptional templates of different base editors and Cre were amplified by PCR using KOD-Plus-Neo (TOYOBO) from plasmids pCMV-BE3 (Addgene#73021), BE4-Gam (Addgene#100806), pCMV-hA3A-BE3-Y130F (Addgene#113428), pCMV-hA3A-eBE3-Y130F (Addgene#113423), xCas9(3.7)-BE3 (Addgene#108380), xCas9(3.7)-BE4 (Addgene#108381) and pZ4f-Cre, purified by the Universal DNA Purification Kit (TIANGEN, Cat#DP214) and then transcribed using the mMACHINE T7 ULTRA Transcription Kit (Invitrogen, Cat#AM1345) following the manufacturer’s instruction. The transcriptional templates of sgRNAs were amplified from pX330-mCherry (Addgene#98750) and transcribed in vitro using the MEGAshortscript T7 kit (Invitrogen, Cat#AM1354) following the manufacturer’s instruction. mRNAs and sgRNAs were subsequently purified with the MEGAclear Transcription Clean-Up Kit (Invitrogen, Cat# AM1908), resuspended in hot (95 °C) RNase-free water and then stored at −80 °C.

pCMV-hA3A-BE3-Y130F used in this study was reported previously [20 (link)] and purchased from Addgene (www.addgene.org/113428, accessed date 20 August 2018). pGL3-U6-sgRNA-PGK-puromycin used in this study [22 (link)] was reported previously and purchased from Addgene (www.addgene.org/51133, accessed date 2 March 2014). SgRNA oligonucleotides targeting the TP53, PTEN, and APC genes (Figure 1a–c) were designed using online software (http://crispor.tefor.net/, accessed date 20 August 2018), and annealed and cloned into the pGL3-U6-sgRNA-PGK-puromycin plasmid after BsaI (NEB, R0535S) digestion. All plasmid DNA was extracted using EndoFree^® Plasmid Maxi Kit (Qiagen, Hilden, Germany).