Data Availability StatementAll the data supporting findings in this manuscript is contained within the manuscript. gene cassette by the cloning-free CRISPR/Cas system with a conventional targeting vector containing 2-kb homology arms . In the present study, we knocked-in a 5-kb TetO-FLEX-hM3Dq/mCherry gene cassette (TetO operator [tetO] sequences followed by inverted Gq-coupled human M3 muscarinic DREADD (designer receptors exclusively activated by designer drug, hM3Dq)/mCherry flanked by two pairs of loxP and loxP2722 [FLEX switch]) to the locus (Fig.?1a) [1, 8, 15, 26]. Since linearization of the donor is IL17RA required for gene cassette knock-in by the PITCh system, we designed a synthetic guide RNA sequence (polyA signal and gcrRNA target sequences (Fig.?1a). The PITCh donor was efficiently digested by in vitro digestion assay (IDA) with crRNA, tracrRNA, and Cas9 protein (Additional file 1: Figure S1). Open in a separate window Fig. 1 Generation of knock-in mice carrying a gene Seliciclib inhibition cassette by the PITCh system. a Targeting strategy for the generation of 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, and crRNAs, tracrRNA and PITCh-donor. The red, purple, and blue boxes Seliciclib inhibition indicate the insert, microhomologies, and target sequences, respectively. c PCR screenings of knock-in newborns. d Summary of 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 We then injected the circular PITCh donor together with chemically synthesized and crRNAs and tracrRNA, and Cas9 protein into one-cell-stage mouse zygotes (Fig.?1b) . We obtained 25 newborns, and screened their tail genomic DNA by PCR with three different primer sets (Fig.?1a) to identify knock-in mice (Fig.?1c, ?,dd Seliciclib inhibition and Additional file 1: Figure S2). We found three knock-in mice Seliciclib inhibition defined by triple PCR positive carrying a TetO-FLEX-hM3Dq/mCherry cassette at the locus (Fig.?1c, ?,dd and Additional file 1: Figure S2). Knock-in efficiency was 12% (Fig.?1d). We also found two partial knock-in mice defined by double PCR positive for LF?+?LR and IF?+?IR carrying a part of the cassette at the locus (mice #10 and #18 in Additional file 1: Figures S2). Next, we sequenced the PCR products of the left and right boundaries between and the TetO-FLEX-hM3Dq/mCherry cassette and found the precise knock-in of the cassette we designed (Fig.?1e). Although left boundaries were precisely knocked-in in two partial knock-in mice, we could not determine their right boundaries (data not shown). We also sequenced the PCR products of non-knock-in alleles amplified with LF and RR primers. These alleles were modified by NHEJ in 92% of the newborn mice (Fig.?1d and Additional file 1: Figure S3). Collectively, the knock-in mice carrying Seliciclib inhibition a gene cassette could be generated by the PITCh system in combination with cloning-free CRISPR/Cas system. However, its efficiency (12%, Fig.?1d) was much lower than that of our previous study (45.5%, ), which was accomplished by the combination of a conventional targeting vector with long homology arms and the cloning-free CRISPR/Cas system, although the length of knock-in cassette in this study was larger than that of previous report (5?kb vs. 2.5?kb). Genetic screening of MMEJ enhancer To enhance the efficiency of the MMEJ-mediated gene cassette knock-in, we conducted genetic screening to identify genes that enhance MMEJ. We constructed a fluorescent reporter system to detect MMEJ-mediated repair of DSBs, similar to the previous report . The reporter plasmid expressing inactive (out-of-frame) EGFP was split by a CRISPR target sequence containing two tandem microhomologies under the control of the CMV promoter (Fig.?2a). When the DSBs in the reporter plasmid induced by CRISPR are repaired through MMEJ between two microhomologies, functional in-frame EGFP is reconstituted and EGFP fluorescence is recovered (Fig.?2a). We chose 13 candidate genes from among those involved in DSB repair pathways ([[[[[[[[dominant-negative [[overexpression increased more than 2.5-fold compared to that of the mock overexpression quantified by imaging analysis (Fig.?2d). The overexpression of did not increase the number of EGFP-positive cells. Conversely, significant reduction of EGFP-positive cells was observed with overexpression compared to that of mock overexpression, consistent with the involvement of this gene in the DSB repair pathway through.