In vitro gRNA Synthesis and CRIS-PITCh Vector Construction

In vitro transcribed gRNAs were prepared according to a previous report (Aida et al., 2015 (link)). Briefly, template DNA fragments were generated using PCR amplification from CRISPR-Cas9 vectors with primers containing a T7 promoter sequence according to a previous protocol (Nakagawa et al., 2016 (link)). Subsequently, the gRNAs were synthesized using a MEGAshortscript T7 Kit (Life Technologies, Carlsbad, CA, USA), and then purified with a MEGAclear Kit (Life Technologies). For the CRIS-PITCh (v2) vector, a genomic region containing Spp1 exons 3 and 4 was amplified from mouse genomic DNA and cloned into pGEM-3Z (Promega, Tokyo, Japan). Subsequently, base substitutions were introduced by site-directed mutagenesis. The two PITCh-gRNA target sites were then added to flank the microhomology sequences. The ssODNs were synthesized by Integrated DNA Technologies (Coralville, IA, USA). The sequences of oligonucleotides for gRNA templates, primers, and ssODNs are listed in Table S3. The recombinant Cas9 protein was obtained from New England Biolabs Japan (Cas9 Nuclease NLS, Streptococcus pyogenes; Tokyo, Japan) or Integrated DNA Technologies Japan (Alt-R™ S.p. Cas9 Nuclease 3NLS; Tokyo, Japan).