Plasmid construction for human cell experiments
The CRISPR/Cas9 vector expressing Cas9 nuclease and an sgRNA targeting EGFP reporter vector was constructed using pX330 vector (Addgene Plasmid #42230) according to the previously described protocol . The all-in-one CRISPR/Cas9 vector expressing Cas9 nuclease and two sgRNAs targeting the FBL or hACTB gene and the donor vector was constructed using the Multiplex CRISPR/Cas9 Assembly System Kit (Addgene Kit #1000000055) as described previously . The oligonucleotide sequences for sgRNA templates were listed in Additional file 1: Table S4.
The overexpression vectors were constructed using an RT-PCR and In-Fusion HD Cloning Kit. Briefly, the full coding sequences for the 14 genes listed in Fig. 2d were amplified using an RT-PCR from total RNAs extracted from HEK293T cells. The amplified cDNAs were cloned into ptCMV-136/63-VR-NG vector (Addgene Plasmid #50700) , replacing the transcription activator-like effector nuclease-coding sequence with each cDNA. The sequences of primers used to construct the overexpression vectors were listed in Additional file 1: Table S4.
The EGFP reporter vector for monitoring MMEJ frequency was constructed using a PCR and In-Fusion HD Cloning Kit (Takara). PITCh(gRNA-s1)-FBL and PITCh(gRNA-s1)-hACTB donor plasmids were constructed using a PCR and TA-cloning with DynaExpress TA PCR Cloning Kit (pTAC-2) (BioDynamics Laboratory Inc.) or TArget Clone -Plus- (Toyobo). The full plasmid sequences of EGFP reporter vector, PITCh(gRNA-s1)-FBL donor vector, and PITCh(gRNA-s1)-hACTB donor vector are shown in Additional file 1: Figure S17.
Cell culture and transfection
HEK293T and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Lipofectamine LTX (Life Technologies) and Opti-MEM (Life Technologies) were used to transfect plasmids, according to the manufacturer’s instructions. Plasmid concentrations, cell numbers, and dishes used were as follows: for the MMEJ-monitoring reporter assay, 100 ng each for all the plasmid vectors (EGFP reporter vector, CRISPR/Cas vector, mCherry vector, and each overexpression vector) into 6 × 104 HEK293T cells using a 96-well plate; for the FACS and DNA sequencing analyses and the imaging quantification using LSM of FBL knocked-in cells, 100 ng each for all the plasmid vectors (PITCh donor vector, CRISPR/Cas vector, and mock, Exo1, or MRE11A vector with or without mCherry vector) into 5.0 × 104 HEK293T cells using a 12-well plate; for the fluorescence observation of FBL knocked-in cells, 50 ng each for all the plasmid vectors (PITCh donor vector, CRISPR/Cas vector, mock or Exo1 vector, and mCherry vector) into 3 × 104 HEK293T cells using a 96-well plate; for gene knock-in at the FBL locus and co-transfection of FBL-mCherry fusion gene, 100 ng each for all the plasmid vectors (PITCh donor vector, CRISPR/Cas vector, and FBL-mCherry vector) into 2.5 × 104 HEK293T cells using a 12-well plate; for the FACS analysis of hACTB knocked-in cells, 100 ng each for all the plasmid vectors (PITCh donor vector, CRISPR/Cas vector, mCherry vector, mock or Exo1 vector, and the vector expressing puromycin resistance gene) into 2 × 105 HeLa cells using a 12-well plate; for the DNA sequencing and fluorescence observation of hACTB knocked-in cells, 8.3–50 ng each for all the plasmid vectors (PITCh donor vector, CRISPR/Cas vector, mCherry vector, mock or Exo1 vector) into 3 × 104 HeLa cells using a 96-well plate; for Western blotting analysis, 2 μg for mock or Exo1 vector into 5 × 104 HEK293T cells using a 6-well plate; for toxicity analysis, 1, 3.5, 12.2, 42.9, and 150 ng each for mock or Exo1 vectors, or 150 ng for zinc-finger nuclease (ZFN) vector, pSTL-ZFA36 , and 150 ng for mCherry vector into 6 × 104 HEK293T cells using a 96-well plate. After transfection, cells were cultured in the growth medium described above for 1 day (reporter assays), 3 days (knock-in experiments and Western blotting), or 2 or 5 days (toxicity analysis), with or without transferring the cells to a larger culture plate. For the FACS analysis of hACTB knocked-in cells, puromycin selection was conducted during 24–72 h post-transfection.
Cells were collected, suspended in PBS, and filtered with a Flowmi Tip Strainer (Bel-Art Products). The number of cells with green (mNeonGreen) and red (mCherry) fluorescence, where needed, was counted using a BD FACS Calibur 4A (BD Biosciences) with a 488-nm laser and the corresponding fluorescence filters. A total of 10,000 cells were recorded for each sample. For toxicity analysis, cell survival rates were determined as the percentages of the number of mCherry-positive cells in total cell counts, as described previously .
For the reporter assays, fluorescence was observed and cell images were captured using a fluorescence microscope (Olympus CKX41) directly in the cultured plates. For the knock-in experiments, cells were moved to collagen-coated glass-bottom 24-well plates at 72 h post-transfection, cultured for additional 24 h, and fixed with 4% paraformaldehyde in PBS. Fluorescence was observed and cell images were captured with a 473-nm and 594-nm lasers using a confocal laser-scanning microscope (Olympus FV-1000D).
Imaging analysis using the ImageJ software
For the reporter assays, the areas containing red fluorescence (mCherry) and green fluorescence (EGFP) were calculated from the captured images using the ImageJ software (https://imagej.nih.gov/ij/). The MMEJ efficiency was determined as the percentages of the EGFP-positive areas in the mCherry-positive areas. For the knock-in experiments, imaging analysis was performed as described in Additional file 1: Figure S4.
Sequencing analysis for human cell experiments
The 5’ and 3’ knock-in junctions of FBL and hACTB genes were amplified by PCR using the primers listed in Additional file 1: Table S4 from the cells collected at 72 h post-transfection without any antibiotic or fluorescence selection. Subsequently, the PCR products were bacterially cloned using the TA-cloning method and sequenced.
Cells were collected and dissolved in sample buffer (final concentrations: 0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.005% bromophenol blue). Western blotting was performed as previously described . Briefly, after denaturation by heating at 95 degree for 10 min, 10 μg of each sample was separated by 4–20% SDS-PAGE (Bio-Rad) along with a protein standard ladder (Nippon Genetics). The samples were then transferred to PVDF membranes. The membranes were cut and blocked in TBS with 0.1% Tween 20 (TBS-T) and 5% skim milk for 1 h at room temperature, then incubated with primary monoclonal antibodies against Exo1 (1:100, Thermo Scientific, Ab-4, clone 266) and β-Actin (1:1000, Santa Cruz Biotechnology, sc-47778) in TBS-T containing 1% skim milk at 4° overnight. Next, the membranes were washed, incubated with HRP-conjugated anti-mouse IgG secondary antibody (1:5000, Jackson ImmunoResearch Laboratories, 715-035-151) at room temperature for 1 h, washed, and visualized with Luminata Forte Western HRP substrate (Millipore). Gel images were taken every 1 min for 20 min using Image Lab software (Bio-Rad). Band intensities of gel images within linear signal range were quantified using Image Lab software and normalized with band intensities of β-Actin.
All research and animal care procedures were approved by the Tokyo Medical and Dental University Animal Care and Use Committee. Mice were housed in groups of 3-5 animals per cage and maintained on a regular 12 h light/dark cycle (8:00–20:00 light period) at a constant 25 °C. Food and water were available ad libitum.
Targeting vector for mouse experiments
Actb-TetO-FLEX-hM3Dq/mCherry PITCh-donor: TetO-FLEX-hM3Dq/mCherry cassette was PCR-amplified from an original plasmid with PrimeSTAR GXL DNA Polymerase (Takara) and primers conjugated with Actb microhomologies and gRNA-s1 crRNA target sequences (Fig. 1a), or with Actb microhomologies (Additional file 1: Figure S13). PCR products were directly used as linear PITCh donors (Additional file 1: Figure S13). Then, PCR products were cloned into plasmids using Mighty TA-cloning Kit (Takara). The PCR products were also inserted into plasmids using In-Fusion HD Cloning Kit (Takara), in some cases.
floxCol12a1 PITCh-donor: The genomic region containing exon 2 of Col12a1 was PCR-amplified as described above with primers conjugated with LoxPs. Then, second PCR was performed with primers conjugated with Col12a1 microhomologies and gRNA-s1 crRNA target sequences. Then, PCR products were cloned into plasmids using Mighty TA-cloning Kit (Takara).
in vitro mRNA transcription
Exo1 mRNA was in vitro transcribed using mMESSAGE mMACHINE T7 ULTRA Kit (Life Technologies). Exo1 mRNA was purified with MEGAclear Kit (Life Technologies) and eluted with Nuclease-free water (Life Technologies). The quality of mRNA was analyzed by NanoDrop (Thermo Scientific) and Bioanalyzer (Agilent Technologies).
The recombinant Cas9 proteins were obtained from Fasmac, New England BioLabs (NEB), and PNA Bio.
Chemical synthesis of crRNA and tracrRNA
crRNAs and tracrRNA (Additional file 1: Table S4) were chemically synthesized and purified by high pressure liquid chromatography (Fasmac).
in vitro digestion assay
IDAs were performed as previously described  with 100 ng of the PITCh-donor vector, gRNA-s1 crRNA, tracrRNA and the Cas9 protein.
Injections were performed as previously described . For Actb-TetO-FLEX-hM3Dq/mCherry knock-in mice, Cas9 proteins, Actb and gRNA-s1 crRNAs, tracrRNA, and PITCh-donor vectors with or without Exo1 mRNA (30 ng/μl, 0.61 pmol/μl, 10 ng/μl, and 10 ng/μl, respectively) were injected into pronuclei of one-cell stage BDF1 zygotes. For linear PCR donor injection, Cas9 proteins, Actb crRNA (and gRNA-s1 crRNA), tracrRNA, and linear PCR PITCh-donor (1.5, 5, or 10 ng/μl) were injected. For floxCol12a1 mice, Cas9 proteins, left and right Col12a1 crRNAs, tracrRNA, and PITCh-donor vectors with Exo1 mRNA (30 ng/μl, 0.61 pmol/μl, 5 ng/μl, and 10 ng/μl, respectively) were injected into pronuclei of one-cell stage C57BL/6 J zygotes. After incubation at 37°, one-cell stage embryos were transferred into pseudopregnant ICR female mice.
PCR screenings of knock-in mice were performed with primers listed in Additional file 1: Table S4 as previously described . PCR products were directly sequenced or cloned, then sequenced. For floxCol12a1 screening, PCR products were digested with HindIII (NEB).
in vitro Cre-recombination
200 ng of PCR products of cloned floxCol12a1 alleles from floxed mice or genomic Col12a1 PCR products of wildtype mice were incubated with Cre recombinase (NEB) and analyzed by 2% agarose gel electrophoresis according to the manufacture’s instruction.
Off-target effects of knock-in mice
The potential off-target candidate loci were analyzed as previously described . PCR primers were listed in Additional file 1: Table S4.
All data are presented as the mean ± SEM. Statistical methods were described in the figure legends for each data set. Statistical significance was set at p < 0.05.