Animals
C57BL/6JJcl mice and F344/Jcl rats were obtained from CLEA Japan Inc., Tokyo, Japan. B6.Cg-Tg(CAG-Cre)CZ-MO2Osb (RBRC01828) and Emx1-cre (RBRC00808) mice were provided by RIKEN BRC (www.en.brc.riken.jp), and W-Tg(CAG-cre)81Jmsk (NBRP-Rat No.0283) rats were from the National Bio Resource Project for the Rat in Japan (www.anim.med.kyoto-u.ac.jp/nbr). The animals were kept under conditions of 50% humidity and a 12:12 h light:dark cycle. They were fed a standard pellet diet (MF, Oriental Yeast Co., Tokyo, Japan) and tap water ad libitum.
Preparation of Cas9, gRNA and lssDNAs
We formally constructed pCas9-polyA and deposited it into the Addgene repository (ID #72602; www.addgene.org/CRISPR). mRNA was transcribed in vitro using a mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies, Carlsbad, CA, USA) from linearized plasmids and was purified using a MEGAClear kit (Life Technologies). To design gRNAs, software tools (www.crispr.genome-engineering.org) predicting unique target sites throughout the mouse and rat genome were used. gRNAs were transcribed in vitro using a MEGAshortscript T7 Transcription Kit (Life Technologies) from synthetic double-strand DNAs obtained from IDT (Integrated DNA Technologies, IA, USA) or Life Technologies.
lssDNAs were prepared by a simple method using nicking endonucleases as we previously reported [11] (Additional file 1: Figure S1). Briefly, double-stranded DNA plasmids comprising of a floxed allele, homology arms, and two nicking endonuclease sites were obtained from Thermo Fisher Scientific (MA, USA) as GeneArt® Gene Synthesis. For digestion, 100 μg of the purified plasmid DNA was incubated at an optimum temperature for 2 to 3 h with nicking endonucleases, such as Nt.BspQI and Nb.BbvCI (New England Biolabs Inc., MA, USA). After purification by ethanol precipitation, the DNAs were denatured with 3-fold amounts of formamide (Nacalai Tesque, Inc., Tokyo, Japan) at 80 °C for 10 min, and then subjected to agarose gel electrophoresis with DynaMarker®Prestain Marker for RNA High concentrations (Biodynamics Laboratory Inc., Tokyo, Japan). Bands corresponding to a single-strand DNA fragment were extracted using NucleoSpin® Gel and PCR Clean-up (Takara Bio, Shiga, Japan). Finally, 2–4 μg of lssDNA was obtained with this method.
Microinjection and electroporation into mouse and rat embryos
Pronuclear-stage mouse embryos were prepared by thawing frozen embryos (CLEA Japan Inc.), or in vitro fertilized embryos, or fresh embryos collected from naturally mated female mice that were superovulated by injection with pregnant mare serum gonadotropin (PMSG: ASKA Animal Health Co., Tokyo, Japan) and human chorionic gonadotropin (HCG: ASKA Animal Health Co.). Rat embryos were collected from 8 to 12 weeks of age females that were superovulated by the administration of 150 U/kg of PMSG followed by 75 U/kg of HCG. After natural mating, pronuclear-stage embryos were collected from the oviducts of the females and cultured in a modified Krebs–Ringer bicarbonate medium or KSOM medium (ARK Resource, Kumamoto, Japan).
In MI, 50 ng/μl Cas9 mRNA, 25 ng/μl gRNA, or 50 ng/μl lssDNA were microinjected into the male pronuclei of embryos using a micromanipulator (Narishige, Tokyo, Japan). In targeting mouse KIAA1322, 30 ng/μl Cas9 protein (PNA Bio, Thousand Oaks, CA, USA) was used instead of Cas9 mRNA [21]. This was cultured in modified Krebs–Ringer bicarbonate or KSOM medium overnight and divided two-cell embryos were transferred into pseudopregnant females.
For EL, 50–100 embryos at 1 h after thawing or 3–4 h after collection were placed into a chamber with 40 μl of serum free media (Opti-MEM, Thermo Fisher Scientific) containing 400 ng/μl Cas9 mRNA, 200 ng/μl gRNA, or 40 ng/μl lssDNA. They were electroporated with a 5 mm gap electrode (CUY505P5 or CUY520P5 Nepa Gene, Chiba, Japan) in a NEPA21 Super Electroporator (Nepa Gene, Chiba, Japan).The poring pulses for the electroporation were voltage 225 V, pulse width 1.5 ms for mouse embryos and 2.0 ms for rat embryos, pulse interval 50 ms, and number of pulses 4. The first and second transfer pulses were voltage 20 V, pulse width 50 ms, pulse interval 50 ms, and number of pulses 5. Mouse or rat embryos that developed to the two-cell stage after the introduction of RNA and lssDNA were transferred into the oviducts of female surrogates anesthetized with isoflurane (DS Pharma Animal Health Co., Ltd., Osaka, Japan).
Genotyping analysis
Genomic DNA was extracted from the tail tip using the KAPA Express Extract DNA Extraction Kit (Kapa Biosystems, London, UK). For PCR and sequence analysis, we used external primers outside the HA, which amplied the targeted region (Additional file 18: Table S4). PCR was performed in a total volume of 15 μl under the following conditions: 1 cycle of 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 60 °C for 1 min and 72 °C for 45 s; and 1 cycle of 72 °C for 3 min. The final reaction mixture contained 200 μM dNTPs, 1.0 mM MgCl2, and 0.66 μM of primer. The PCR products were then directly sequenced using the BigDye Terminator v3.1 cycle sequencing mix and the standard protocol for an Applied Biosystems 3130 DNA Sequencer (Life Technologies). To confirm mosaic mutations, we sequenced individual TA clones in some cases.