Generation of Percc1 Knockout Mice by CRISPR/Cas9

Percc1 knockout mice were generated by CRISPR/Cas9 editing as previously described^{26 (link),27 (link)}. Single guide RNAs (sgRNAs) were constructed using 60-mer oligonucleotides and an sgRNA cloning vector (Addgene plasmid 41824)^{28 (link)} according to the following protocol: http://www.addgene.org/static/cms/files/hCRISPR_gRNA_Synthesis.pdf. The sgRNA target site sequences are provided in Supp. Table S9. Cas9 mRNA was generated using a human codon-optimized Cas9 gene from plasmid pDD921^{29 (link)}. T7-promoter-Cas9-polyA and T7-promoter-sgRNA amplicons were PCR amplified from pDD921 and sgRNA clones, respectively, using Phusion polymerase (New England Biolabs). Cas9 RNA was generated by in vitro transcription from the T7promoter-Cas9-polyA amplicon using mMESSAGE mMACHINE T7 Kit (ThermoFisher Scientific), following the manufacturer’s instructions. sgRNA RNA was generated by in vitro transcription from the T7-promoter-sgRNA amplicon using MEGAshortscript Kit (ThermoFisher Scientific), following manufacturer’s instructions. In vitro-transcribed RNA was cleaned using MEGAclear Kit (ThermoFisher Scientific), following the manufacturer’s instructions. RNA was eluted into RNase-free microinjection buffer (10 mM Tris, pH7.5; 0.1 mM EDTA). The RNA was then assessed by electrophoresis on a 10% TBE Urea PAGE gel.