CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

All mouse experiments were approved by the Institutional Animal Care and Use Committee of the University of Tokyo (approval number PA17-63) and Osaka University (approval number Biken-AP-H30-01) and performed according to their guidelines as well as the ARRIVE guidelines (https://arriveguidelines.org).

1. Targeting vector construction

1. Amplify a KI cassette (CreERT here, template DNA for the PCR is originally from a commercial gene synthesis company) and an approximate 1 kb fragment of 5'- or 3'-homology arms by PCR. For DNA cloning (step 1.3), add 15-mer overlap sequences at the 5' end of each PCR primer. Purify the PCR DNA fragments by agarose gel electrophoresis, followed by extraction of the DNA fragment using a DNA purification kit.
2. Linearize the backbone plasmid (e.g., pUC19 or pBS) using an appropriate restriction enzyme(s) that uniquely digests somewhere in the multi-cloning site for cloning. Then, purify the digested plasmid using a DNA purification kit.
3. Clone each PCR fragment (e.g., 5'-homology arm, 3'-homology arm) and KI sequence simultaneously into the digested plasmid using a DNA cloning kit according to the manufacturer's instructions.
4. Transform the competent cells using the constructed plasmid (step 1.3) by heating at 42 °C for 1 min in a water bath, then seed them on an LB plate containing the appropriate concentration of antibiotics such as ampicillin or kanamycin. Culture them overnight for resistant clone selections.
5. Pick up several (four to eight) individual colonies using 200 µL pipette tips and transfer the tips into 3 mL of liquid LB containing appropriate antibiotics (100 µg/mL ampicillin or 20 µL/mL kanamycin) and culture overnight at 37 °C with shaking.
6. Purify the plasmid the following day using an endotoxin-free grade commercial plasmid purification kit according to the manufacturer's instructions. Confirm the cloned sequence in the plasmid by Sanger sequencing. Adjust the plasmid concentration at 1 µg DNA/µL using nuclease-free water. Use the plasmid as the gene targeting vector.

2. Preparation of the mouse embryonic fibroblast (MEF) as feeder cells for ESC

1. Sacrifice 8-10-week-old pregnant ICR female mice (14.5 days postcoitum) by cervical dislocation. Wipe the abdomen well by using 70% (v/v) ethanol for sterilization, cut the abdomen using orbital scissors and forceps, and recover the fetus-containing uterus.
2. Place the uterus into a 100 mm dish containing 10 mL of phosphate bovine serum (PBS) containing 100 U/mL penicillin and 100 µg/mL streptomycin. Remove the placentas and extraembryonic tissues from the fetuses using orbital scissors and forceps. Mince the fetuses using orbital scissors and transfer the PBS solution containing the minced fetal cells to a 50 mL conical tube.
3. Centrifuge at 280 x g for 5 min at 4 °C and discard the supernatant by aspiration. Add 10 mL of 0.25% (w/v) trypsin-EDTA solution, mix well by pipetting, and then incubate for 10 min at 37 °C using a water bath for trypsin digestion.
4. Add 20 mL of MEF medium (DMEM containing 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin) to stop the enzymatic reaction, mix well by gentle inversion, and centrifuge at 280 x g for 5 min at 4 °C. Discard the supernatant by aspiration, add 10 mL of MEF medium, and mix well by pipetting.
5. Seed the cell suspension in several new 100 mm dishes (prepare the same number of dishes as the number of fetuses with 10 mL of MEF medium for one 100 mm dish). Change the medium 4 to 5 h post-seeding to remove unattached cells and let the attached MEF grow. Passage the cells when they reach ~80% confluency using trypsin.
6. Place the culture dishes containing the proliferating MEF in MEF medium, approximately 12-15 days post-seeding, in an X-ray irradiating device. Expose the MEF with a 50 Gy X-ray (total) to halt the cell cycle according to the manufacturer's instruction.
7. Trypsinize the irradiated MEF by adding 2 mL of 0.25% trypsin-EDTA for one 100 mm dish for 5 min at 37 °C, then add 4 mL of MEF medium to stop trypsin digestion and collect the cell suspension in a new 50 mL tube.
8. Wash the MEF with a fresh MEF medium by centrifugation at 280 x g for 5 min at 4 °C, count the number of cells using a cell counter with trypan blue staining, and freeze them at 1.6 x 10⁶ cells/tube in a cell-freezing medium. Store until use; cells can be stably stored in liquid nitrogen for several years.

3. Cas9-RNP-DNA mixture preparation

1. Dissolve tracrRNA and crisprRNA in dilution buffer (each RNA concentration at 200 µM) by pipetting. Mix the tracrRNA solution, crisprRNA solution, and dilution buffer (volume ratio at 2:2:5) by gentle tapping and anneal each RNA using a thermal cycler at 95 °C for 10 min followed by 1 °C/min stepdown cycles until the temperature reaches 4 °C.
   NOTE: The gRNA sequence was designed using CRISPOR, http://crispor.tefor.net.
2. Incubate the annealed RNAs, 10 µg/µL Cas9 nuclease, and the electroporation buffer mixed in a volume ratio of 5:3:2 at 37 °C for 20 min to form the Cas9-RNP complex. In routine work, mix, and incubate 1.25 µL of annealed RNA, 0.75 µL of Cas9 nuclease, and 0.5 µL of the electroporation buffer for one locus genome editing.
3. Mix the following materials in a 1.5 mL tube: 10 µL of electroporation buffer, 1 µL of Cas9-RNP complex (step 3.2), and 1 µL of circular targeting vector (1 µg/µL, step 1.6). The total amount of Cas9-RNP-DNA mixture is 12 µL. Keep the Cas9-RNP-DNA mixture on ice until electroporation.
   ​NOTE: To prevent re-cleavage of the gRNA after KI, design the gRNA in a position where the gRNA recognition sequence is split by the integration of the KI donor. If the gRNA cannot be designed in such a location, several nucleotide silent mutations, by which the amino acid sequence does not change, are included in the plasmid of the KI donor.

4. Gene targeting of ESCs

1. To prepare the gelatin-coated cell culture dishes, add 0.1% (w/v) gelatin solution at 2 mL for a 60 mm dish, 500 µL for a 24-well plate, 100 µL for a 96-well plate, and incubate for 2 h in a humid incubator. Remove the gelatin solution, wash twice with the same amount of PBS, and store at room temperature until use.
2. Thaw mitotically inactivated frozen MEF stock (step 2.2) using a 37 °C water bath for 1 min, and place the MEF on a gelatin-coated 60 mm dish (one frozen stock tube of MEF containing 1.6 x 10⁶ cells for two 60 mm dishes, step 2.2) 1 day before ESC seeding.
3. Thaw a frozen ESC stock tube (stored at 2 x 10⁵ ESCs/tube; cell counting was carried out using a cell counter) using a 37 °C water bath for 1 min, and place 1 x 10⁵ ESCs onto a 60 mm dish containing pre-seeded MEF (step 4.1).
4. Culture in 4 mL ESC culture medium (ESCM, DMEM-based modified medium with 15% (v/v) fetal bovine serum, 2 mM L-glutamine substrate, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.1 mM 2-mercaptoethanol, leukemia inhibitory factor, and t2i (0.2 µM PD0325901 and 3 µM CHIR99021) which maintains pluripotency of ESC¹¹) at 37 °C with 5% CO₂ until the ESC reaches 50% to 70% confluency.
   NOTE: We use three different lines of ESC: JM8.A3 from B6N, V6.5 from B6-129 F1, and in-house developed BALB/c ESC. The same KI protocols in this manuscript are used for all ESC lines.
5. Wash the culturing ESC with 4 mL of PBS, then treat with 800 µL of 0.25% trypsin-EDTA solution for 5 min at 37 °C for digestion. Add 1 mL of ESCM to inactivate the trypsin and dissociate the ESCs to single cells by pipetting.
6. Centrifuge the ESC-containing solution for 5 min at 280 x g, discard the supernatant, resuspend ESCs in fresh 1 mL of ESCM, and count the number of cells using a cell counter with trypan blue staining. Transfer 1 x 10⁵ ESCs to a new 1.5 mL tube and wash them three times with PBS by centrifugation at 500 x g, 4 °C.
7. Resuspend the ESC pellet in 12 µL of Cas9-RNP-DNA mixture (step 3.3) and mix well by gentle pipetting to avoid bubbling. Serve the resuspended ESC for electroporation. The electroporation system uses a single pulse at 1,400 V and 30 ms for Cas9-RNP-DNA transduction (Figure 1).
8. Culture the electroporated ESCs in a 60 mm dish containing 4 mL of ESCM with mitotically inactivated MEF (section 4.2). Change the medium every day.
9. Wash the ESCs with 4 mL of PBS 3 to 5 days after the electroporation, then treat with 800 µL of 0.25% trypsin-EDTA and culture at 37 °C for 5 min in a humid incubator for digestion. Add 2 mL of ESCM to stop the trypsin digestion and centrifuge the cell mixture at 280 x g for 5 min.
10. Discard the supernatant, resuspend the ESCs in 1 mL of fresh ESCM, and count the cell concentration using a cell counter with trypan blue staining. Passage the ESCs at 1 x 10³ ESCs per 60 mm dish containing ESCM and feeder MEF. Change the medium every day until the colony picks-up.
    ​NOTE: The reason why the ESC is passed once before pick-up was that genome editing may continue to occur after ESC division, so the first colony is not always the clone in many cases. Therefore, the first passage of ESC before the colony pick-up is an essential step to pick-up single clones.

5. PCR genotyping of targeted ESCs

1. Aspirate ESCM from the ESC culture dish 5 to 7 days after the first passage (step 4.9) and add 4 mL of PBS. Pick up single ESC colonies with 5 µL of PBS using a 20 µL pipette under a stereomicroscope (30x to 40x magnification). Place the individual colonies in a well of a round-bottom 96-well plate containing 15 µL of 0.25% trypsin-EDTA solution.
2. Keep the 96-well plate on ice until 48 individual colonies are picked-up. Incubate the 96-well plate for 5 min in a 37 °C humid 5% CO₂ incubator, then add 80 µL of ESCM to stop trypsin digestion and dissociate ESC colonies into single cells by pipetting.
   NOTE: Since the KI efficiency of this method is usually 10%-50%, we routinely pick up 48 clones and first perform genotyping using 24 of them. If multiple KI clones cannot be obtained at this point, we further screen for KI using the remaining 24 clones.
3. Transfer 40 µL of ESC suspension (step 5) to a gelatin-coated feeder-free 96-well plate containing 50 µL of ESCM per well for PCR genotyping. Transfer the remaining 60 µL of ESC suspension into a well in a gelatin-coated 24-well plate, which contains 500 µL of ESCM and feeder MEF, to make the frozen ESC stock.
4. Stock individual ESC clones cultured in the 24-well plate (step 5.3) until they will reach 60% to 80% confluency. Recover individual ESC clones by trypsinization as mentioned above, collect cells in a new 1.5 mL tube, and centrifuge at 280 x g for 5 min. Discard the supernatant, resuspend the cells in 500 µL of cell freezing medium, and freeze them using a -80 °C deep freezer.
5. For PCR genotyping, culture ESC clones in the 96-well feeder-free plate (step 5.3) until the ESC reaches more than 90% confluency. Remove ESCM from each well by aspiration and wash twice with 100 µL of PBS.
6. Aspirate the PBS and add 100 µL of lysis buffer containing proteinase K, mix well, and transfer the ESC lysate to a new 1.5 mL tube. Heat the ESC lysate at 65 °C for at least 1 h using a heat chamber. Extract and purify the genomic DNA using a conventional phenol-chloroform DNA purification method followed by ethanol precipitation.
7. Dissolve the precipitated DNA in 20 µL of DNase-free water and determine the purity and concentration of DNA using a spectrophotometer. Perform genomic PCR using locus-specific primer sets to amplify the target region. Then, check the sequence and choose the ESC clones with the desired KI sequences.

6. Preparation of eight-cell stage embryo and microinjection of ESCs

1. Inject 5 IU of pregnant mare serum gonadotrophin (PMSG) intraperitoneally into adult ICR females. After 48 h, inject 5 IU of human chorionic gonadotrophin (hCG) into the same females, then mate the hormone-treated females with the ICR males. Check the copulatory plug in the vagina the following day (embryonic day 0.5, ED0.5).
   NOTE: To evaluate chimerism by coat color ratios, use blastocyst from the ICR strain as a recipient for a ESC that has black or agouti coat color background, or blastocyst from the C57BL/6 strain as a recipient for a ESC that has the albino background.
2. Collect the oviduct at ED1.5 and place it in HEPES buffered medium (FHM) drops. Recover the two-cell or four-cell stage embryos by flushing the oviduct with FHM using a flushing needle inserted into the infundibulum.
3. Wash the collected embryos by moving them into several fresh FHM drops using a mouth pipette. Culture the embryos in 50 µL of KSOM drop on a 35 mm cell culture dish covered with mineral oil at 37 °C, 5% CO₂ incubator for 1 day until the eight-cell or morula stage (ED2.5) is reached.
4. If the embryos are not used on the next collection day, freeze them by vitrification according to the CARD protocol (http://card.medic.kumamoto-u.ac.jp/card/english/sigen/manual/ebvitri.html), and store them in liquid nitrogen until use.
5. Prepare glass pipettes for holding the embryos (80-100 µm outer diameter) and ESC injection (about 13 µm diameter) using a puller and microforge.
6. Integrate a holding pipette that contains FHM into a capillary holder connected to a microinjector and set up on the left side of the micromanipulator. Connect an injection pipette to another microinjector and set it up on the right side. Align the two pipettes straight in the field of the view of microscope.
7. Dispense 5 µL drops of 12% (w/v) polyvinylpyrrolidone (PVP) in FHM medium and 5 µL drops of FHM onto the same lid of a 60 mm dish side-by-side and cover the drops with mineral oil. Transfer embryos in the 5 µL FHM drop and add 1-5 µL of the ESC suspension to the FHM drop that contains embryos.
   NOTE: Leave the ESC and MEF suspension for 30 min in the 4 mL ESM on 60mm culture dish before the drop preparation. Since MEFs stick to the bottom faster than ESCs, this 30-minute incubation allows for the concentration of unattached ES cells in the supernatant.
8. Wash inside the injection pipette with PVP, then move to the drop containing the embryos and ESCs.
9. Pick up three individual ECS doublets that have just finished their cell division (six cells) in the injection pipette. Hold an eight-cell or morula stage embryo, which has completed compaction, with the holding pipette by aspiration. Make a hole in the zona pellucida with a piezoelectric pulse (intensity: 3; speed: 3). Expel the six-celled ESC inside the zona pellucida and pull the pipette out of the embryos.
10. Wash the ESC-injected embryos with several KSOM drops gently using a mouth pipette and incubate them in a new 50 µL KSOM drop covered with mineral oil at 37 °C, 5% CO₂ until the embryos develop to the blastocyst stage.
11. Transfer the 10 ESC-injected blastocysts into each uterine horn of pseudopregnant female mice (2.5 days postcoitum, 20 blastocysts per head).
12. After the surrogate mother has given birth, select at least two male chimeras with the highest chimerism ratio (70% or higher as confirmed by coat color), and then mate them with appropriate strain females to obtain F1 heterozygous KI.
13. Breed individual KI mice with appropriate partners to obtain mice with desirable genotype(s) or genetic backgrounds suitable for research.