Efficient Gene Editing using TALEN and CRISPR/Cas9

RNAs for TALENs and CRISPR/Cas9 were prepared as described previously [5 (link)21 (link)27 (link)]. Briefly, TALEN and Cas9 mRNAs were synthesized in vitro from linear DNA templates (ToolGen, Korea) via the mMESSAGE mMACHINE T7 Ultra kit (Invitrogen, USA) according to the manufacturer's instructions. DNA templates for sgRNAs were synthesized in vitro by PCR. The sgRNAs were produced from these templates using a MEGA-shortscript T7 kit (Invitrogen, USA) according to the manufacturer's instructions. The sgRNAs were diluted in RNase-free injection buffer (0.25 mM EDTA, 10 mM Tris at pH 7.4) and introduced into the cytoplasm of fertilized eggs by microinjection. TALEN target sites were as follows: TALEN #1 for p16, 5′-TGCATGACGT GCGGGCACTG-3′; TALEN #2 for p16, 5′-TTCGGGG CGTTGGGCGAAAC-3′ for both B6 and FVB mice; TALEN #1 of p19, 5′-TTCGTGCGATCCCGGAGACC-3′; TALEN #2 of p19, 5′-TCACGAAAGCCAGAGCGC AG-3′ for both B6 and FVB mice. The following sequences were used for sgRNA synthesis: sgRNA #1 of p27, 5′-GCGGATGGACGCCAGACAAG-3′; sgRNA #2 of p27, 5′-GGACTTGGAGAAGCACTGCC-3′.