C57bl 6n mice

The experimental protocols were approved by the Wako Animal Experiment Committee of RIKEN. Kmt2c heterozygous mutant mice were maintained by breeding Kmt2c +/fs mice with wildtype C57BL6/N mice (CLEA Japan, Inc.) in a 12 h:12 h light-dark cycle. The mice for behavioral tests were obtained by in vitro fertilization using sperms of the heterozygous mutant mice and eggs derived from C57BL6/N mice (CLEA Japan, Inc.). Genotyping of all mutant mice was performed using genomic DNA derived from tail tips as previously reported [18] . Briefly, tail biopsies were conducted on postnatal days 14-21, and each tail tip was incubated in 100 μL of lysis buffer (25 mM NaOH, 0.2 mM EDTA) at 95 °C for 20-30 min. An equal volume of 40 mM TRIZMA hydrochloride was added to the lysate after incubation and the mixture was vortexed briefly. Afterward, 1 μL of the mixture was diluted with water 2.5-fold and was PCR-amplified under the conditions shown in Supplementary Table 1. The PCR products derived from wildtype and mutant allelles were discriminated by agarose gel electrophoresis or Sanger sequencing by an ABI 3730xl sequencer (Life Technologies, CA, USA) using BigDye Terminator V3.1 (Thermo Fisher Scientific, MA, USA) (Supplementary Table 1).

To obtain single guide RNA (sgRNA) for the recognition of the Kmt2c locus, oligomers were first generated by annealing of the following primers: forward primer, 5′-CACCGGAATGGACCACCACCTCGAG-3′, and the reverse primer, 5′-AAACCTCGAGGTGGTGGTCCATTCC-3′. The oligomers were integrated into the BbsI site downstream of the U6 promoter of the pSpCas9(BB)-2A-GFP vector (PX458) (Addgene, MA, USA). This integration step followed our previous reports [17, 18] . After the integration, the sgRNA for the microinjection into the fertilized eggs was transcribed by MEGAshortscript Kit (Life Technologies, Carlsbad, CA, USA) using the generated sgRNA-SpCas9-GFP all-in-one vector as a template. A mixture of sgRNA (50 ng/μl) and Cas9 mRNA (100 ng/μl, Merck Millipore, MA, USA) was microinjected into the cytoplasm of fertilized eggs obtained from C57BL6/N mice (CLEA Japan, Inc., Tokyo, Japan). The injected eggs were transferred into the oviducts of pseudopregnant ICR female mice. Whole exome sequencing was performed to screen for possible off-target mutations using genomic DNA purified from tail samples of F1 mutant mice with the GenElute Mammalian Miniprep kit (Merck Millipore). We validated the introduction of novel insertion/deletion of nucleotides after the filtering as previously reported [18] .