Generation of Cas 9 mRNA and PLCD1 sgRNA

The proper target sequence of PLCD1 sgRNAs was acquired from Optimized CRISPR Design (http://crispr.mit.edu). Generation of PLCD1 sgRNAs and Cas 9 mRNA was performed as described [28]. pUC57-sgRNA expression vector was purchased from Addgene (Plasmid 51132). Complimentary oligonucleotides containing the PLCD1 sgRNA target sequences were annealed and cloned into the Bsa I site of pUC57-sgRNA. These recombination plasmids were then sequenced to screen correct insertion of the target sequences. pUC57-sgRNA-PLCD1 linearized by Dra I was purified, followed by in vitro transcription using MEGAshortscript™ Kit (Ambion). Cas9 mRNA was synthesized using the mMessagemMachine T7 Ultra Kit (Ambion), followed by purification. The quality of Cas9 mRNA and sgRNAs was confirmed by agarose gel electrophoresis.