CRISPR-Cas9 Reagent Production and Delivery

Production and purification of Cas9 mRNA were performed as described previously (Yoshimi et al. 2014 (link), 2016 (link)). Cas9 protein was obtained from IDT (Alt-R S.p. Cas9 Nuclease V3, Integrated DNA Technologies, IA, USA). sgRNAs were designed using an online program (https://crispor.tefor.net/) to predict unique target sites throughout the mouse and rat genome. Single-guide RNAs were transcribed in vitro from synthetic double-strand DNAs obtained from IDT or Thermo Fisher Scientific using a MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific, MA, USA). Specific crRNAs were purchased from IDT (Alt-R CRISPR-Cas9 crRNA) and were assembled with a tracrRNA (Alt-R CRISPR-Cas9 tracrRNA) before use according to the instructions of the manufacturer. Several plasmids used as knock-in donors were purchased from Thermo Fisher Scientific (GeneArt Gene Synthesis). In accordance with the conventional methods, all plasmids were transformed into Escherichia coli and extracted with NucleoSpin Plasmid Transfection grade (MACHEREY–NAGEL Gmbh & Co. KG, Germany).