Fig. 1

Generation of knock-in mice carrying a gene cassette by the PITCh system. a Targeting strategy for the generation of Actb-TetO-FLEX-hM3Dq/mCherry knock-in mice by the PITCh system. Purple highlights indicate microhomologies between endogenous Actb locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Actb and gRNA-s1 crRNAs, tracrRNA and PITCh-donor. The red, purple, and blue boxes indicate the insert, Actb microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of knock-in newborns. d Summary of Actb-TetO-FLEX-hM3Dq/mCherry knock-in mouse production by the PITCh system. e Sequences of boundaries between Actb and TetO-FLEX- hM3Dq/mCherry cassette. Blue characters indicate microhomologies. IF: internal forward primer, IR: internal reverse primer, LF: left forward primer, LR: left reverse primer, RF: right forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, WT: wildtype, KI: knock-in, WPRE: woodchuck hepatitis virus posttranscriptional regulatory element, pA: polyA, and KI/+: tail genomic DNA of F1 heterozygous knock-in pup derived from #13 (KI#2) F0 knock-in mouse