Animal experiments
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 three to five animals per cage and maintained on a regular 12 hours light/dark cycle (8:00 to 20:00 light period) at a constant 25°C. Food and water were available ad libitum.
CRISPR/Cas plasmid
A pair of oligo DNAs (Hokkaido Systems Science, Sapporo, Hokkaido, Japan) corresponding to Actb sgRNA was hybridized and ligated using Quick Ligase (New England BioLabs (NEB), Ipswich, MA, USA) into linearized pX330 plasmid (Addgene, 42230; Feng Zhang, MIT) digested with BbsI (NEB) as previously described [18,19]. Oligo DNAs and primers are listed in Table S6 in Additional file 1.
Targeting vector
The pActb-TetO-FLEX-EGFP-polyA targeting vector was constructed based on a pAAV-TetO-FLEX-HA-mKate2-TeNT-polyA plasmid (a gift from Akihiro Yamanaka, Nagoya University) with several modifications. First, HA-mKate2-TeNT was excised by digestion with XhoI (NEB) and HindIII (NEB), and replaced with PCR amplified inverted EGFP. Second, AAV2-ITR was excised by digestion with NarI (NEB) and BstEII (NEB), and replaced with a PCR-amplified 2.0 kb Actb fragment from C57BL/6 J mouse genomic DNA for left homology arm using a In-Fusion HD Cloning Kit (Takara, Otsu, Shiga, Japan). Finally, the PCR-amplified 2.0 kb Actb fragment for right homology arm was inserted into the plasmid digested with NotI (NEB) and MluI (NEB) by In-Fusion reaction.
Single-strand annealing assay
SSA assay using HEK293T cells was performed as described previously [43]. Briefly, Actb-pX330 or empty pX330 plasmids, firefly luciferase reporter vector containing the PCR-amplified Actb target sequence (Table S6 in Additional file 1), and renilla luciferase-expressing reference vector were co-transfected into HEK293T cells in a 96-well plate using Lipofectamine LTX (Life Technologies, Grand Island, NY, USA). At 24 hours post-transfection, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
Cel-I assay in Neuro2A cells
Cel-I assay using Mouse Neuro2A cells was performed as described previously [43]. Briefly, Actb-pX330 or empty pX330 plasmids was transfected into Neuro2A cells in a 6-well plate using Lipofectamine LTX (Life Technologies). After 72 hours post-transfection, genomic DNA was isolated using a DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA). Then, Actb loci were PCR amplified from the purified genomic DNA with primers (Table S6 in Additional file 1). PCR products were denatured, digested at 42°C for 30 minutes with a Surveyor Mutation Detection Kit (Transgenomic, Omaha, NE, USA), and analyzed by electrophoresis in 3% agarose gel stained with ethidium bromide. Gel images were obtained with a ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA) and analyzed by Image Lab software (Bio-Rad).
In vitro RNA transcription
Cas9 mRNA and Actb-sgRNA were prepared as described previously [5]. Cas9 and Actb-sgRNA were PCR amplified from Actb-pX330 with T7 promoter-attached primers (Table S6 in Additional file 1). T7-Cas9 and T7-Actb-sgRNA PCR products were purified with a PCR Purification Kit (Qiagen) and used as the template for in vitro transcription using a mMESSAGE mMACHINE T7 ULTRA Kit (Life Technologies) and MEGAshortscript T7 Kit (Life Technologies). Cas9 mRNA and Actb-sgRNA were purified with a MEGAclear Kit (Life Technologies) and eluted with Nuclease-free water (Life Technologies). The quality of RNAs was analyzed using a NanoDrop (Thermo Scientific, Waltham, MA, USA) and Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Cas9 proteins
The recombinant Cas9 proteins were obtained from NEB and PNA Bio (Thousand Oaks, CA, USA).
Chemical synthesis of crRNA and tracrRNA
Actb-crRNA (5′-cauuaugaguccuuaagugaGUUUUAGAGCUAUGCUGUUUUG-3′) and -tracrRNA (5′- AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3′) were designed with some modification of previously reported methods [12,19], and chemically synthesized and purified by polyacrylamide gel electrophoresis (Fasmac, Atsugi, Kanagawa, Japan).
In vitro digestion assay
Cas9 proteins (30 ng/μl) and chemically synthesized Actb-crRNA (8.7 ng/μl) and -tracrRNA (14.3 ng/μl), or in vitro transcribed Actb-sgRNA (25 ng/μl) were incubated with Actb target PCR products (30 ng/μl) in a Cas9 Nuclease Reaction Buffer (NEB) at 37°C for 60 minutes as previously described [13], then treated with RNase A (5 mg) and incubated at 37°C for 30 minutes to remove RNA [44]. Reactions were stopped with 6× DNA loading buffer containing 30% glycerol, 1.2% SDS and 250 mM EDTA, and analyzed by electrophoresis in 2% agarose gel as described above.
Injection
For knockout mouse production, Cas9 mRNAs and Actb-sgRNA were diluted and mixed in 0.1 TE buffer (10 mM Tris-HCl, 0.1 mM EDTA (pH 8.0)) to a working concentration of 50 and 20 ng/μl, respectively. One-cell-stage zygotes were obtained by mating of BDF1 males and females (CLEA Japan, Meguro, Tokyo, Japan), and then frozen and stored until use. The mixture of Cas9 mRNAs and Actb-sgRNA was injected into the cytoplasm using a micromanipulator and microscope (Leica, Wetzlar, Germany) and injector (Eppendorf, Hauppauge, NY, USA). After incubation at 37°C for 24 hours, two-cell-stage embryos were transferred into pseudopregnant ICR female mice (CLEA Japan).
For knock-in mouse production by RNA injection, Cas9 mRNAs, Actb-sgRNA, and pActb-TetO-FLEX-EGFP-polyA were diluted and mixed in 0.1 TE buffer to a working concentration of 5, 2.5, and 10 ng/μl, respectively, as previously described [5]. The injection mixture was injected into pronuclei of one-cell-stage zygotes.
For knock-in mouse production by protein injection, Cas9 proteins, Actb-sgRNA, or Actb-crRNA and tracrRNA, and pActb-TetO-FLEX-EGFP-polyA were diluted and mixed in 0.1 TE buffer to a working concentration of 30 or 100 ng/μl, 2.5 or 25 ng/μl, or 0.061 or 0.61 pmol/μl, and 10 ng/μl, respectively. The mixture was incubated at 37°C for at least 15 minutes, and then injected into pronuclei of one-cell-stage zygotes.
PCR screening
Genomic DNA was prepared from F0 and F1 newborn tails by proteinase K treatment and a subsequent standard phenol extraction method as described previously [45,46]. Knock-in mice were screened by PCR with ExTaq (Takara) and three different pairs of primers (Table S6 in Additional file 1) and analyzed by electrophoresis in 1 or 2% agarose gel as described above. PCR products were further cloned with TOPO TA Cloning Kit (Life Technologies) and analyzed by sequencing.
Southern blotting
Southern probe (0.7 kb) was PCR amplified (Primers: Table S6 in Additional file 1) from BDF1 genomic DNA and cloned with a TOPO TA Cloning Kit, and DIG-labeled with a DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche, Penzberg, Upper Bavaria, Germany). The genomic DNA was digested with HindIII and separated on 0.7% agarose gel, transferred to nylon membranes, positively charged (Roche), hybridized with DIG-labeled probe, and detected with CDP-Star (Roche) as previously described [47].
Off-target effects
The potential off-target candidate loci containing up to 3 bp mismatches compared with the 20 bp guide sequence of Actb sgRNA were predicted by the CRISPR design tool [18,25,48]. The off-target candidate loci were amplified by PCR using primers listed in Table S6 in Additional file 1 and analyzed by direct sequencing as previously described [47].
Primary fibroblast cultures
The ear tips derived from 2-week-old mice were diced into small pieces, incubated at 37°C for 30 minutes with 4 mg/ml collagenase L (Nitta Gelatin, Naniwa, Osaka, Japan) and 4 mg/ml dispase (Life Technologies), and then cultured with 10% fetal bovine serum/Dulbecco’s modified eagle medium at 37°C and 10% CO2 for several days. pCAG-Cre, pCMV-tTA (Takara), and pCMV-DsRed (Takara) were co-transfected into primary fibroblast cells in a six-well plate using Lipofectamine LTX with Plus reagent (Life Technologies). Images were acquired on a FV500 confocal microscope and Fluoview software (Olympus, Shinjuku, Tokyo, Japan).
Statistical analyses
All data are presented as the mean ± standard error of the mean. Statistical methods were described in the figure legends for each data set. Briefly, Student’s t-tests were used to compare differences between any two groups. One-way ANOVA with post hoc Tukey-Kramer tests were used to compare differences between three groups. Statistical significance was set at P < 0.05.