We thank Saki Nishioka in Osaka University, Biotechnology Research and Development (nonprofit organization), and Mio Kikuchi and Reiko Sakamoto in the Institute of Medical Science, The University of Tokyo, for their excellent technical assistance. This work was supported by: the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS) KAKENHI grants to MI (JP19H05750, JP21H05033), and MO (20H03162); the Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST) grant to MI (JPMJCR21N1 ); the Eunice Kennedy Shriver National Institute of Child Health and Human Development to MI (R01HD088412); the Bill &#38; Melinda Gates Foundation to MI (Grand Challenges Explorations grant INV-001902); and the Grant for Joint Research Project of the Research Institute for Microbial Diseases, Osaka University to MI, and MO.

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
<ol>
	<li>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&#39;- or 3&#39;-homology arms by PCR. For DNA cloning (step 1.3), add 15-mer overlap sequences at the 5&#39; 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.</li>
	<li>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.</li>
	<li>Clone each PCR fragment (e.g., 5&#39;-homology arm, 3&#39;-homology arm) and KI sequence simultaneously into the digested plasmid using a DNA cloning kit according to the manufacturer&#39;s instructions.</li>
	<li>Transform the competent cells using the constructed plasmid (step 1.3) by heating at 42 &#176;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.</li>
	<li>Pick up several (four to eight) individual colonies using 200 &#181;L pipette tips and transfer the tips into 3 mL of liquid LB containing appropriate antibiotics (100 &#181;g/mL ampicillin or 20 &#181;L/mL kanamycin) and culture overnight at 37 &#176;C with shaking.</li>
	<li>Purify the plasmid the following day using an endotoxin-free grade commercial plasmid purification kit according to the manufacturer&#39;s instructions. Confirm the cloned sequence in the plasmid by Sanger sequencing. Adjust the plasmid concentration at 1 &#181;g DNA/&#181;L using nuclease-free water. Use the plasmid as the gene targeting vector.</li>
</ol>
2. Preparation of the mouse embryonic fibroblast (MEF) as feeder cells for ESC
<ol>
	<li>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.</li>
	<li>Place the uterus into a 100 mm dish containing 10 mL of phosphate bovine serum (PBS) containing 100 U/mL penicillin and 100 &#181;g/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.</li>
	<li>Centrifuge at 280 x g for 5 min at 4 &#176;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 &#176;C using a water bath for trypsin digestion.</li>
	<li>Add 20 mL of MEF medium (DMEM containing 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin) to stop the enzymatic reaction, mix well by gentle inversion, and centrifuge at 280 x g for 5 min at 4 &#176;C. Discard the supernatant by aspiration, add 10 mL of MEF medium, and mix well by pipetting.</li>
	<li>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.</li>
	<li>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&#39;s instruction.</li>
	<li>Trypsinize the irradiated MEF by adding 2 mL of 0.25% trypsin-EDTA for one 100 mm dish for 5 min at 37 &#176;C, then add 4 mL of MEF medium to stop trypsin digestion and collect the cell suspension in a new 50 mL tube.</li>
	<li>Wash the MEF with a fresh MEF medium by centrifugation at 280 x g for 5 min at 4 &#176;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.</li>
</ol>
3. Cas9-RNP-DNA mixture preparation
<ol>
	<li>Dissolve tracrRNA and crisprRNA in dilution buffer (each RNA concentration at 200 &#181;M) 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 &#176;C for 10 min followed by 1 &#176;C/min stepdown cycles until the temperature reaches 4 &#176;C. 
	NOTE: The gRNA sequence was designed using CRISPOR,&#160;http://crispor.tefor.net.</li>
	<li>Incubate the annealed RNAs, 10 &#181;g/&#181;L Cas9 nuclease, and the electroporation buffer mixed in a volume ratio of 5:3:2 at 37 &#176;C for 20 min to form the Cas9-RNP complex. In routine work, mix, and incubate 1.25 &#181;L of annealed RNA, 0.75 &#181;L of Cas9 nuclease, and 0.5 &#181;L of the electroporation buffer for one locus genome editing.</li>
	<li>Mix the following materials in a 1.5 mL tube: 10 &#181;L of electroporation buffer, 1 &#181;L of Cas9-RNP complex (step 3.2), and 1 &#181;L of circular targeting vector (1 &#181;g/&#181;L, step 1.6). The total amount of Cas9-RNP-DNA mixture is 12 &#181;L. Keep the Cas9-RNP-DNA mixture on ice until electroporation. 
	&#8203;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.</li>
</ol>
4. Gene targeting of ESCs
<ol>
	<li>To prepare the gelatin-coated cell culture dishes, add 0.1% (w/v) gelatin solution at 2 mL for a 60 mm dish, 500 &#181;L for a 24-well plate, 100 &#181;L 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.</li>
	<li>Thaw mitotically inactivated frozen MEF stock (step 2.2) using a 37 &#176;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.</li>
	<li>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 &#176;C water bath for 1 min, and place 1 x 105 ESCs onto a 60 mm dish containing pre-seeded MEF (step 4.1).</li>
	<li>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 &#181;g/mL streptomycin, 0.1 mM 2-mercaptoethanol, leukemia inhibitory factor, and t2i (0.2 &#181;M PD0325901 and 3 &#181;M CHIR99021) which maintains pluripotency of ESC11) at 37 &#176;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.</li>
	<li>Wash the culturing ESC with 4 mL of PBS, then treat with 800 &#181;L of 0.25% trypsin-EDTA solution for 5 min at 37 &#176;C for digestion. Add 1 mL of ESCM to inactivate the trypsin and dissociate the ESCs to single cells by pipetting.</li>
	<li>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 &#176;C.</li>
	<li>Resuspend the ESC pellet in 12 &#181;L 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).</li>
	<li>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.</li>
	<li>Wash the ESCs with 4 mL of PBS 3 to 5 days after the electroporation, then treat with 800 &#181;L of 0.25% trypsin-EDTA and culture at 37 &#176;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.</li>
	<li>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. 
	&#8203;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.</li>
</ol>
5. PCR genotyping of targeted ESCs
<ol>
	<li>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 &#181;L of PBS using a 20 &#181;L pipette under a stereomicroscope (30x to 40x magnification). Place the individual colonies in a well of a round-bottom 96-well plate containing 15 &#181;L of 0.25% trypsin-EDTA solution.</li>
	<li>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 &#176;C humid 5% CO2 incubator, then add 80 &#181;L 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.</li>
	<li>Transfer 40 &#181;L of ESC suspension (step 5) to a gelatin-coated feeder-free 96-well plate containing 50 &#181;L of ESCM per well for PCR genotyping. Transfer the remaining 60 &#181;L of ESC suspension into a well in a gelatin-coated 24-well plate, which contains 500 &#181;L of ESCM and feeder MEF, to make the frozen ESC stock.</li>
	<li>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 &#181;L of cell freezing medium, and freeze them using a -80 &#176;C deep freezer.</li>
	<li>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 &#181;L of PBS.</li>
	<li>Aspirate the PBS and add 100 &#181;L 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 &#176;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.</li>
	<li>Dissolve the precipitated DNA in 20 &#181;L 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.</li>
</ol>
6. Preparation of eight-cell stage embryo and microinjection of ESCs
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Wash the collected embryos by moving them into several fresh FHM drops using a mouth pipette. Culture the embryos in 50 &#181;L of KSOM drop on a 35 mm cell culture dish covered with mineral oil at 37 &#176;C, 5% CO2 incubator for 1 day until the eight-cell or morula stage (ED2.5) is reached.</li>
	<li>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.</li>
	<li>Prepare glass pipettes for holding the embryos (80-100 &#181;m outer diameter) and ESC injection (about 13 &#181;m diameter) using a puller and microforge.</li>
	<li>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.</li>
	<li>Dispense 5 &#181;L drops of 12% (w/v) polyvinylpyrrolidone (PVP) in FHM medium and 5 &#181;L 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 &#181;L FHM drop and add 1-5 &#181;L 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.</li>
	<li>Wash inside the injection pipette with PVP, then move to the drop containing the embryos and ESCs.</li>
	<li>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.</li>
	<li>Wash the ESC-injected embryos with several KSOM drops gently using a mouth pipette and incubate them in a new 50 &#181;L KSOM drop covered with mineral oil at 37 &#176;C, 5% CO2 until the embryos develop to the blastocyst stage.</li>
	<li>Transfer the 10 ESC-injected blastocysts into each uterine horn of pseudopregnant female mice (2.5 days postcoitum, 20 blastocysts per head).</li>
	<li>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.</li>
	<li>Breed individual KI mice with appropriate partners to obtain mice with desirable genotype(s) or genetic backgrounds suitable for research.</li>
</ol>

<ol><li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337 (6096), 816-821 (2012).</li>
<li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((One-step+generation+of+mice+carrying+mutations+in+multiple+genes+by+CRISPR%2FCas-mediated+genome+engineering%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.</a> Cell. 153 (4), 910-918 (2013).</li>
<li>Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((One-step+generation+of+mice+carrying+reporter+and+conditional+alleles+by+CRISPR%2FCas-mediated+genome+engineering%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.</a> Cell. 154 (6), 1370-1379 (2013).</li>
<li>Oji, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR%2FCas9+mediated+genome+editing+in+ES+cells+and+its+application+for+chimeric+analysis+in+mice%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice.</a> Scientific Reports. 6, 31666(2016).</li>
<li>Kaneko, T., Mashimo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simple+genome+editing+of+rodent+intact+embryos+by+electroporation%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Simple genome editing of rodent intact embryos by electroporation.</a> PLoS One. 10 (11), 0142755(2015).</li>
<li>Chen, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-frequency+genome+editing+using+ssDNA+oligonucleotides+with+zinc-finger+nucleases%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases.</a> Nature Methods. 8 (9), 753-755 (2011).</li>
<li>Hashimoto, M., Yamashita, Y., Takemoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electroporation+of+Cas9+protein%2FsgRNA+into+early+pronuclear+zygotes+generates+non-mosaic+mutants+in+the+mouse%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse.</a> Developmental Biology. 418 (1), 1-9 (2016).</li>
<li>Mansour, S. L., Thomas, K. R., Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disruption+of+the+proto-oncogene+int-2+in+mouse+embryo-derived+stem+cells%3A+a+general+strategy+for+targeting+mutations+to+non-selectable+genes%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes.</a> Nature. 336 (6197), 348-352 (1988).</li>
<li>Johnson, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+of+nonexpressed+genes+in+embryonic+stem+cells+via+homologous+recombination%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Targeting of nonexpressed genes in embryonic stem cells via homologous recombination.</a> Science. 245 (4923), 1234-1236 (1989).</li>
<li>Hasty, P., Ramires-Solis, R., Krumlauf, R., Bradley, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Introduction+of+a+subtle+mutation+into+the+Hox-2.6+locus+in+embryonic+stem+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Introduction of a subtle mutation into the Hox-2.6 locus in embryonic stem cells.</a> Nature. 350 (6315), 243-246 (1991).</li>
<li>Ozawa, M., Emori, C., Ikawa, M. Gene targeting in mouse embryonic stem cells via CRISPR/Cas9 ribonucleoprotein (RNP) mediated genome editing. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aOzawa%2C+M+%22Genome+Editing+in+Animals+-+Methods+and+Protocols+2nd+edition%22">Genome Editing in Animals - Methods and Protocols 2nd edition</a>. Hatada, I. , (2022).</li>
<li>Yagi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Derivation+of+ground-state+female+ES+cells+maintaining+gamete-derived+DNA+methylation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Derivation of ground-state female ES cells maintaining gamete-derived DNA methylation.</a> Nature. 548 (7666), 224-227 (2017).</li>
<li>Gassmann, M., Donoho, G., Berg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maintenance+of+an+extrachromosomal+plasmid+vector+in+mouse+embryonic+stem+cells%5BTitle%5D)+AND+%22Proceedings+of+National+Academy+of+Sciences%22%5BJournal%5D)">Maintenance of an extrachromosomal plasmid vector in mouse embryonic stem cells.</a> Proceedings of National Academy of Sciences. 92 (5), 1292-1296 (1995).</li>
<li>Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+mouse+induced+pluripotent+stem+cells+without+viral+vectors%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Generation of mouse induced pluripotent stem cells without viral vectors.</a> Science. 322 (5903), 949-953 (2008).</li>
<li>Poueymirou, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((F0+generation+mice+fully+derived+from+gene-targeted+embryonic+stem+cells+allowing+immediate+phenotypic+analyses%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses.</a> Nature Biotechnology. 25 (1), 91-99 (2007).</li>
<li>Hu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+production+of+chimeric+mice+from+embryonic+stem+cells+injected+into+4-+to+8-cell+and+blastocyst+embryos%5BTitle%5D)+AND+%22Journal+of+Animal+Science+and+Biotechnology%22%5BJournal%5D)">Efficient production of chimeric mice from embryonic stem cells injected into 4- to 8-cell and blastocyst embryos.</a> Journal of Animal Science and Biotechnology. 4 (1), 1-7 (2013).</li>
<li>Guo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contribution+of+Mouse+Embryonic+Stem+Cells+and+Induced+Pluripotent+Stem+Cells+to+Chimeras+through+Injection+and+Coculture+of+Embryos%5BTitle%5D)+AND+%22Stem+Cells+International%22%5BJournal%5D)">Contribution of Mouse Embryonic Stem Cells and Induced Pluripotent Stem Cells to Chimeras through Injection and Coculture of Embryos.</a> Stem Cells International. 2014, 409021(2014).</li>
<li>Bodai, Z., Bishop, A. L., Gantz, V. M., Komor, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+double-strand+break+indel+byproducts+with+secondary+guide+RNAs+improves+Cas9+HDR-mediated+genome+editing+efficiencies%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies.</a> Nature Communications. 13 (1), 2351(2022).</li>
<li>Kobayashi, T., Goto, T., Oikawa, M., Sanbo, M., Yoshida, F., Terada, R., Niizeki, N., Kajitani, N., Kazuki, K., Kazuki, Y., Hochi, S., Nakauchi, H., Surani, A., Hirabayashi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blastocyst+complementation+using+Prdm14-deficient+rats+enables+efficient+germline+transmission+and+generation+of+functional+mouse+spermatids+in+rats%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Blastocyst complementation using Prdm14-deficient rats enables efficient germline transmission and generation of functional mouse spermatids in rats.</a> Nature Communications. 12 (1), 1328(2021).</li>
<li>Miura, K., Matoba, S., Hirose, M., Ogura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+chimeric+mice+with+spermatozoa+fully+derived+from+embryonic+stem+cells+using+a+triple-target+CRISPR+method+for+Nanos3%5BTitle%5D)+AND+%22Biology+of+Reproduction%22%5BJournal%5D)">Generation of chimeric mice with spermatozoa fully derived from embryonic stem cells using a triple-target CRISPR method for Nanos3.</a> Biology of Reproduction. 104 (1), 223-233 (2021).</li>
</ol>

The authors have no competing interests to disclose.

Gene targeting of ESCs followed by chimera production has been conventionally used for developing gene-manipulated mice. Nevertheless, gene knock-in efficiency remains low even though the targeting vector contains long (&#62; several kb in usual) homology arms with positive or negative drug selection gene cassettes. Our protocol introduced a tuned-up ESC KI method of long exogenous DNA using a circular plasmid without any drug selection cassettes as a targeting vector accompanied by Cas9-RNP-mediated genome editing at an acceptable efficiency for routine works. Thus, this protocol could help to substantially reduce the amount of time taken in producing genetically modified chimeric mice compared with the conventional ESC targeting.
In this protocol, we used CRISPR/Cas9-RNP to induce site-specific double-strand breaks in the genome, and a circular plasmid was used as the targeting vector instead of a linearized one. Linearized plasmids are conventionally used as a targeting vector for gene KI because of their increased efficiency of genomic integration8,9,10. However, many genomic integrations are nonspecific even though the vector contains homologous arms9. On the other hand, circular plasmids are rarely used as targeting vectors because of their hard-to-integrate feature into the genome of ESCs12 or fibroblasts13. Thus, it would be possible that applying a circular plasmid as a targeting vector accompanied by CRISPR/Cas9-mediated genome editing minimizes nonspecific random integration but maximizes the site-specific integration into the ESC genome. It would be notable that the induction efficiency of the double-strand break by CRISPR/Cas9 is quite important14, thus it would be essential to use gRNA which induces a double-strand break at high efficiency. It should be considered to re-design gRNA when the KI efficiency is too low. In the case shown in Figure 2, 40.9% of ESC clones showed a KI-specific band. In fact, as the core lab of the institute, we routinely perform gene targeting using ESCs by the method described here and have achieved KI efficiencies of 10%-50% for 1-2 kb sequences such as Cre, CreERT, or fluorescent reporters, although there are some variations depending on the gene locus. The longest DNA sequences we have successfully KI using this method is about 11.2 kb into the Rosa26 locus, and the efficiency was 12.2% (five KI colonies out of 41 analyzed colonies).
It would also be of note that this protocol uses eight-cell or morula stage embryos as recipients for ESC microinjection but not blastocyst stage embryos. Although we have not conducted comparing experiments in this paper, several reports have shown that injection of ESCs into eight-cell or morula stage embryos significantly improves the efficiency of ESC contribution to chimeric offspring compared to the ESC injection into blastocysts15,16,17,18. Indeed, ESC injections of various ESC lines, including C57BL/6, B6-129 F1, and BALB/c, commonly resulted in high coat color chimera development in some, but not all, offspring (see Figure 4). The limitation of the method is that the ESCs injected into the preimplantation embryo could not always contribute to germ cells in a chimera19,20. Germline transmission of ESCs would depend on the quality of each ESC clone19. Therefore, it would be advantageous to develop multiple ESC clone lines as a backup for stable chimeric mouse production. In conclusion, the protocols presented here use simple targeting vectors which include only each homology arm and gene of interest for KI without any drug-resistant cassette. Therefore, the vector construction would be much easier, and the culture period could be shortened compared to the conventional ESC targeting technique. This would help in quickly and facilitatively producing various types of genetically modified mice for future analysis in life science.

Producing genetically modified mice and analyzing their phenotype enables us to understand specific gene functions in detail, in vivo. Numerous important findings have been uncovered using gene-modified animal models in the life science field. Furthermore, since the report of genome editing technology using CRISPR/Cas91, research using genetically modified mice has quickly spread to many laboratories2,3. Genome editing of mouse zygotes by CRISPR/Cas9 has achieved acceptable efficiency for developing short DNA modification, such as indel mutation-oriented gene knockout4, single nucleotide replacement, or short peptide-tag insertion using single-stranded oligonucleotides (ssODNs) as knock-in (KI) donors5. On the other hand, the KI of large DNA fragments into zygotes by genome editing remains at a low efficiency compared to the short-size DNA modification6,7. In addition, it is difficult to use mouse strains such as BALB/c, which is an important strain for specific research areas like immunology, for zygote-based genome editing because their preimplantation embryos are susceptible to in vitro manipulation.
Another way to develop genetically modified mouse models is to use the embryonic stem cell (ESC) targeting technique followed by ESC injection into the preimplantation embryo to produce chimeras8,9,10, which is still routinely used as a conventional method. Although the acquisition rate for obtaining accurate KI-ESC clones is not very high in conventional ESC targeting methods, ESC targeting offers some advantages compared to zygote genome editing, especially for long DNA KI. For example, the KI efficiency of long DNA fragments (&#62; several kb) into the zygote genome is less evident6,7, and many zygotes are needed to develop even one line of KI mouse, which is undesirable in the current perspective of animal experiments. In contrast to zygote genome editing, long DNA targeting to ESCs followed by chimera production needs significantly fewer embryos than zygote genome editing. Furthermore, even though the preimplantation embryos from BALB/c are susceptible to in vitro manipulation, their ESCs can be maintained and handled in vitro11 as other competent 129 or F1 background ESCs, therefore, applicable for chimera productions. However, even though a targeting vector contains 5&#39; and 3&#39; homologous arms and drug resistance gene cassettes for positive or negative selection, the conventional KI efficiency of ESCs is generally insufficient, because of the high frequency of random genomic integration8,10, Thus, an improved method with precise ESC targeting efficiency is required. Recently, we reported a tuned-up ESC KI method using CRISPR/Cas9-based genome editing to achieve higher KI efficiency than conventional targeting methods11. The method we describe here is based on this procedure which enables long DNA (&#62; several to 10 kb) KI to ESCs with acceptable efficiency for routine works without drug selection; thus, the vector construction procedure would be much easier and need a shorter period, or the cell culture period would also become significantly shorter.

<table><tbody><tr><td>BALB/c ESC</td><td>-</td><td>-</td><td>ESC developed from BALB/c strain</td></tr><tr><td>Bambanker</td><td>Nippon Genetics</td><td>CS-02-001</td><td>Cell-freezeing medium. Section 2.6 and elsewhere</td></tr><tr><td>Cas9 Nuclease V3</td><td>IDT</td><td>1081059</td><td>Section 3.2 and elsewhere.</td></tr><tr><td>CHIR99021</td><td>FUJIFILM Wako</td><td>038-23101</td><td>Section 4.3</td></tr><tr><td>CreERT gene fragment</td><td>GeneWiz</td><td></td><td>Section 1.1.</td></tr><tr><td>CRISPR-Cas9 crisprRNA</td><td>IDT</td><td></td><td>crisprRNA. Section 3.1 and elsewhere.</td></tr><tr><td>CRISPR-Cas9 tracrRNA</td><td>IDT</td><td>1072534</td><td>tracrRNA. Section 3.1 and elsewhere.</td></tr><tr><td>DMEM</td><td>Nacalai</td><td>08458-45</td><td>MEF medium. Section 2.3 and elsewhere</td></tr><tr><td>Duplex buffer</td><td>IDT</td><td>1072534</td><td>RNA dilution buffer. Section 3.1 and elsewhere.</td></tr><tr><td>FastGene Gel/PCR Extraction Kit</td><td>Nippon Genetics</td><td>FG-91302</td><td>Section 1.1 and 1.2.</td></tr><tr><td>GlutaMax</td><td>Thermo Fisher</td><td>35-050-061</td><td>L-glutatime substrate</td></tr><tr><td>hCG</td><td>ASKA Animal Health</td><td></td><td>Section 6.1.</td></tr><tr><td>In-Fusion HD Cloning Kit</td><td>Clontech</td><td>639648</td><td>DNA cloning kit. Section 1.3 and elsewhere</td></tr><tr><td>JM8.A3 ESC</td><td>EuMMCR</td><td>-</td><td>ESC developed from C57BL/6N strain</td></tr><tr><td>Knock-out DMEM</td><td>Thermo Fisher</td><td>10829018</td><td>Section 4.3 and elsewhere, DMEM-based modified commercial medium.</td></tr><tr><td>KSOM</td><td>Merck</td><td>MR-121-D</td><td>Section 6.3 and 6.9.</td></tr><tr><td>Leukemia inhibitory factor</td><td>FUJIFILM Wako</td><td>125-05603</td><td>Section 4.3. No unit concentration data is supplied by the provider. Used 1,000-fold dilution in this protocol.</td></tr><tr><td>Neon Electroporation system</td><td>Thermo Fisher</td><td>MPK5000</td><td>Section 3.2, 4.5 and elsewhere. The system containes electroporation buffer as well used in section 3.2.</td></tr><tr><td>NucleoSpin Plasmid Transfection-grade</td><td>Takara</td><td>U0490B</td><td>Section 1.6.</td></tr><tr><td>PD0325901</td><td>FUJIFILM Wako</td><td>162-25291</td><td>Section 4.3</td></tr><tr><td>PMSG</td><td>ASKA Animal Health</td><td></td><td>Section 6.1.</td></tr><tr><td>Tail lysis buffer</td><td>Nacalai</td><td>06169-95</td><td>Section 5.5.</td></tr><tr><td>Trypsin-EDTA</td><td>Nacalai</td><td>32777-15</td><td>Section 2.2 and elsewhere</td></tr><tr><td>V6.5 ESC</td><td>-</td><td>-</td><td>ESC developed from B6J-129 F1 strain</td></tr><tr><td>X-ray irradiation device</td><td>Hitachi</td><td>MBR-1618R-BE</td><td>Section 2.6.</td></tr></tbody></table>

crispr/cas9-mediated highly efficient gene targeting in embryonic stem cells for developing gene-manipulated mouse models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

We have targeted specific gene(s) in the ESC followed by chimera production to develop gene-manipulated mouse production according to our previous manuscript11. ESC genotyping (described in section 4) is routinely carried out by PCR using primers. The primers are designed on genomic sequences outside the homology arms and the specific sequences in the KI DNA fragment (Figure 2A). In that case, no wild-type allele is amplified, whereas a PCR amplicon of a specific size is detected only when the targeted exogenous DNA is KI at the target locus. Representative genotyping PCR results are shown in Figure 2B. Nine out of 22 clones (40.9%) showed a KI-specific band in this case. Three representative targeting results, including the result shown in Figure 2, are shown in Table 1. These results indicate that the method shown here is efficient and reproducible for gene KI without any drug selections.
ESC is picked up in an injection pipette, then a hole in the zona pellucida is made using a piezoelectric plus, and the ESC is released between the embryonic cells (Figure 3). Technically, this protocol to inject an ESC into an eight-cell or morula stage embryo is similar to the protocol for ESC injection into a blastocyst commonly used in many mouse facilities. Representative chimeric mice are shown in Figure 4. For coat color evaluation of chimerism, the embryo from the ICR strain (albino, white hair) was used as a recipient of B6 or B6-129 F1 ESC, and B6 embryos were used as a recipient of BALB/c ESCs (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64385/64385fig01.jpg" /> 
Figure 1: A schematic of CRISPR/Cas9 ribonucleoprotein (RNP)-mediated circular plasmid integration into the specific locus of an ESC genome. Electroporation introduces a circular plasmid as a targeting vector into ESCs with Cas9-RNP. Abbreviations: GOI = gene of interest, HA = homology arm. <a href="https://www.jove.com/files/ftp_upload/64385/64385fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64385/64385fig02.jpg" /> 
Figure 2: Genomic PCR analyses for KI screening of targeted ESC clones. (A) KI-specific PCR primers are designed on genomic sequences outside the homology arms (forward) and in the specific sequences in the KI DNA fragment (reverse). (B) Representative genotyping PCR results. A wild-type genome was used as a negative control. Abbreviations: GOI = gene of interest, HA = homology arm. <a href="https://www.jove.com/files/ftp_upload/64385/64385fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64385/64385fig03.jpg" /> 
Figure 3: Representative images of eight-cell stage embryo injection. (A) For microinjection, three doublets of ESCs (six cells, arrowheads) are picked up. (B) A hole in the zona pellucida is made with a piezoelectric pulse, and ESCs are expelled in-between each blastomere. Scale bar shown is 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64385/64385fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64385/64385fig04.jpg" /> 
Figure 4: Representative images of chimera mice. (A,B) The ESC derived from C57BL/6N (JM8.A3, Agouti hair; A) or B6-129 F1 (Agouti hair; B) are injected into ICR (albino, white hair) embryos, followed by transferring the embryos to the mother surrogates. (C) The ESC derived from BALB/c (albino, white hair) is injected into C57BL/6J (black hair) embryos, followed by transferring the embryos to mother surrogates. <a href="https://www.jove.com/files/ftp_upload/64385/64385fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Project ID</td>
			<td>Choromosome</td>
			<td>5&#39;HA length (bp)</td>
			<td>3&#39;HA length (bp)</td>
			<td>Insert size (bp)</td>
			<td>Number of analyzed clones</td>
			<td>Number of KI clones</td>
			<td>Efficiency (%)</td>
			<td>Remarks</td>
		</tr>
		<tr>
			<td>R26-CC*</td>
			<td>Chr6</td>
			<td>965</td>
			<td>1006</td>
			<td>5321</td>
			<td>23</td>
			<td>2</td>
			<td>8.7%</td>
			<td>-</td>
		</tr>
		<tr>
			<td>R4-03*</td>
			<td>Chr8</td>
			<td>1000</td>
			<td>997</td>
			<td>3070</td>
			<td>22</td>
			<td>6</td>
			<td>27.3%</td>
			<td>-</td>
		</tr>
		<tr>
			<td>P4-01*</td>
			<td>Chr15</td>
			<td>1000</td>
			<td>1000</td>
			<td>2569</td>
			<td>22</td>
			<td>9</td>
			<td>40.9%</td>
			<td>Shown in Figure 2</td>
		</tr>
	</tbody>
</table>
Table 1: KI efficiencies in three independent genomic loci in the ESC. *These projects have not been published so far. The name of these genes will be disclosed in independent manuscripts in the future.

Watch this Scientific Journal Video about CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models at JoVE.com

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

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...

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Developmental Biology

3.8K Views.  The University of Tokyo. Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

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

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

Genetics

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus AAV and 2-Cell Embryo Electroporation

This protocol presents an optimized approach for producing genetically modified rat models. Adeno-associated virus (AAV) is used to deliver a DNA repair template, and electroporation is used to deliver CRISPR-Cas9 reagents to complete the genome editing process in 2-cell embryo.

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal experiments. When generating animal models, the electroporation of zygotes offers higher efficiency, simplicity, cost, and throughput as an alternative to the gold standard method of microinjection. Electroporation is also gentler, with higher viability, and reliably delivers Cas9/single-guide RNA (sgRNA) ribonucleoproteins (RNPs) into the zygotes of common laboratory mouse strains (e.g., C57BL/6J and C57BL/6N) that approaches 100% delivery efficiency. This technique enables insertion/deletion (indels) mutations, point mutations, the deletion of whole genes or exons, and small insertions in the range of 100-200 bp to insert LoxP or short tags like FLAG, HA, or V5. While constantly being improved, here we present the current state of CRISPR-EZ in a protocol that includes sgRNA production through in vitro transcription, embryo processing, RNP assembly, electroporation, and the genotyping of preimplantation embryos. A graduate-level researcher with minimal experience manipulating embryos can obtain genetically edited embryos in less than 1 week using this protocol. Here, we offer a straightforward, low-cost, efficient, high-capacity method that could be used with mouse embryos.

Here, we describe a simple technique intended for the efficient generation of genetically modified mice called CRISPR RNP Electroporation of Zygotes (CRISPR-EZ). This method delivers editing reagents by electroporation into embryos at an efficiency approaching 100%. This protocol is effective for point mutations, small genomic insertions, and deletions in mammalian embryos.

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal ...

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Immunology and Infection

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...