Name: use of freeze-thawed embryos for high-efficiency production of genetically modified mice
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We wish to thank Hitomi Sawada and Elizabeth Garcia for animal care. This work was supported by KAKENHI (15K20134, 17K11222, 16H06276 and 16K01946) and Hokugin Research Grant (to H.N.), and Jichi Medical University Young Investigator Award (to H.U.).&#160;The Otsuka Toshimi Scholarship Foundation supported M.D.

All animal care and procedures performed in this study were undertaken according to the rules and regulations of the Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Animal Care Committee of Laboratory Animals of University of Toyama, University of Tokyo, Jichi University, and Max Planck Florida Institute for Neuroscience. Information about all reagents is showed in the Table of Materials.
1. CRISPR reagents design
<ol>
	<li>crR...

<ol>
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The described protocol allows for the generation of GM mice with high efficiency and low mosaic rates (Table 1). It enables researchers without advanced embryo manipulation skills to create mutant mice easily because it takes advantage of the latest and most useful advances in both reproductive engineering and genome editing technologies: CRISPR/Cas9 ribonucleoprotein (RNP) and electroporation into freeze-thawed embryos. These advances facilitated and expedited the generation of the GM mice. As described...

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a scientific breakthrough that provides unprecedented targeted modification in the genome1. The CRISPR/Cas9 system is comprised of Cas9 protein and guide RNA (gRNA) with two molecular components: a target-specific CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA)2 . A gRNA directs the Cas9 protein to the specific locus in the genome, 20 nucleotides complementary to crRNA, and adjacent to the protospacer adjacent motif (PAM). The Cas9 protein binds to the target sequence and induces double-strand breaks (DSBs) that are repaired by either error-prone nonhomologous end joining (NHEJ) or high fidelity homology-directed repair (HDR)3,4,5. The NHEJ leads to insertions or/and deletions (indels), and hence to gene loss of function when a coding sequence is targeted. The HDR leads to precise genome editing in the presence of a repair template containing homology sequences3,4,5. The NHEJ and HDR have been harnessed to generate knockout and knock-in mice, respectively.
While the CRISPR/Cas9 system has markedly accelerated the generation of GM mice with outstanding efficacy and fidelity, scientists who apply these methods often encounter technical challenges. First, conventional protocols require microinjection for introducing the CRISPR editing tools into the pronucleus of fertilized eggs6,7. This technique is time-consuming and usually requires extensive training. Thus, several groups replaced microinjection with electroporation8,9,10,11,12,13. However, in the early electroporation protocols fresh embryos were used for electroporation. This caused another problem, because preparing fresh embryos before each experiment is difficult14.
Recently we and others have combined the use of freeze-thawed embryos and electroporation for genome editing, which facilitates the generation of GM mice15,16. This protocol enables researchers without advanced embryo manipulation skills to rapidly generate animal models of human diseases with high efficiency. The protocol also significantly reduces practical challenges in generating GM mice, such as genetic heterogeneity in the founders16. To overcome mosaicism, we perform the electroporation of CRISPR reagents within 1 h after embryo thawing to ensure that editing occurs before the first replication of the genome. Another improvement includes the use of Cas9 protein instead of Cas9 mRNA to reduce undesirable mosaicism17. Furthermore, we developed an optimal method for one-cell embryo cryopreservation that increases the developmental rate to the two-cell stage16: use of fetal bovine serum (FBS) dramatically improves the survival of freeze-thawed oocytes after fertilization, perhaps by the same mechanism that makes freeze-thawed unfertilized oocytes more resilient18.
Here we present a comprehensive protocol for the generation of GM mice using freeze-thawed embryos, including the modified method for cryopreservation of one-cell C57BL/6J embryos. It includes 1) gRNA design, CRISPR reagent preparation and assembly; 2) IVF, cryopreservation, and thawing of one-cell embryos; 3) Electroporation of CRISPR reagents into freeze-thawed embryos; 4) Embryo transfer into the oviduct of pseudopregnant female mice; and 5) Genotyping and sequence analysis of the F0 founder animals.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25 M Sucrose</td>
			<td>ARK Resource Co., Ltd. (Kumamoto, Japan)</td>
			<td>SUCROSE</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M DMSO</td>
			<td>ARK Resource Co., Ltd. (Kumamoto, Japan)</td>
			<td>1M DMSO</td>
			<td></td>
		</tr>
		<tr>
			<td>Butorphanol</td>
			<td>Meiji Seika Pharma Co., Ltd. (Tokyo, Japan)</td>
			<td>Vetorphale 5mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Cas9 protein: Alt-R&#174; S.p. HiFi Cas9 Nuclease 3NLS</td>
			<td>Integrated DNA Technologies, Inc. (Coralville, IA)</td>
			<td>1081060</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Japan SLC (Hamamatsu, Japan)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>DAP213</td>
			<td>ARK Resource Co., Ltd. (Kumamoto, Japan)</td>
			<td>DAP213</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich, Inc. (St. Louis, MO)</td>
			<td>ES-009-C</td>
			<td></td>
		</tr>
		<tr>
			<td>hCG</td>
			<td>MOCHIDA PHARMACEUTICAL CO., LTD (Tokyo, Japan)</td>
			<td>HCG Mochida 3000</td>
			<td></td>
		</tr>
		<tr>
			<td>HTF</td>
			<td>ARK Resource Co., Ltd. (Kumamoto, Japan)</td>
			<td>HTF</td>
			<td></td>
		</tr>
		<tr>
			<td>ICR mice</td>
			<td>Japan SLC (Hamamatsu, Japan)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Petterson Vet Supply, Inc. (Greeley, CO)</td>
			<td>07-893-1389</td>
			<td></td>
		</tr>
		<tr>
			<td>KSOM</td>
			<td>ARK Resource Co., Ltd. (Kumamoto, Japan)</td>
			<td>KSOM</td>
			<td></td>
		</tr>
		<tr>
			<td>LN2 Tank</td>
			<td>Chart Industries (Ball Ground, GA)</td>
			<td>XC 34/18</td>
			<td></td>
		</tr>
		<tr>
			<td>M2</td>
			<td>ARK Resource Co., Ltd. (Kumamoto, Japan)</td>
			<td>M2</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Nippon Zenyaku Kogyo Co.,Ltd. (Koriyama, Japan)</td>
			<td>1124401A1060</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon Co. (Tokyo, Japan)</td>
			<td>SMZ745T</td>
			<td></td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Sandoz K.K. (Tokyo, Japan)</td>
			<td>1124401A1060</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease free buffer</td>
			<td>Integrated DNA Technologies, Inc. (Coralville, IA)</td>
			<td>1072570</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin DNA extraction kit</td>
			<td>Takara Bio Inc (Kusatsu, Japan)</td>
			<td>740952 .5</td>
			<td></td>
		</tr>
		<tr>
			<td>One-hole slide glass</td>
			<td>Matsunami Glass Ind., Ltd. (Kishiwada, Japan)</td>
			<td>S339929</td>
			<td></td>
		</tr>
		<tr>
			<td>One-step type Electroporator</td>
			<td>BEX Co., Ltd. (Tokyo, Japan)</td>
			<td>CUY21EDIT II</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Liquid</td>
			<td>NACALAI TESQUE Inc. (Kyoto, Japan)</td>
			<td>SP 26137-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum plate electrode</td>
			<td>BEX Co., Ltd. (Tokyo, Japan)</td>
			<td>LF501PT1-10, GE-101</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>ASKA Animal Health Co., Ltd (Tokyo, Japan)</td>
			<td>SEROTROPIN 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone iodide</td>
			<td>Professional Disposables International, Inc. (Orangeburg, NY)</td>
			<td>C12400</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced-Serum Minimal Essential Medium: OptiMEM I</td>
			<td>Sigma-Aldrich, Inc. (St. Louis, MO)</td>
			<td>22600134</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-step type Electroporator</td>
			<td>Nepa Gene Co., Ltd. (Ichikawa, Japan)</td>
			<td>NEPA21</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Our modified method for cryopreservation of one-cell embryos, including incubation in HTF containing 20% FBS for 10 min followed by cryopreservation in 1 M DMSO and DAP213 solution, improved the developmental rate of the freeze-thawed embryos into the two-cell stage (Figure 1, p = 0.009, Student&#8217;s t-test).The freeze-thawed embryos were used for the production of GM mice and electroporation conditions were optimized: five repeats of 25 V with 3 ms pulses and 97 ms intervals using an ele...

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

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. ...

Our protocol facilitates the preparation of genetically modified mice easily, efficiently, and rapidly from single cell mouse embryos. Our protocol combine the use of freeze-thawed one-cell stage embryos and electroporation allowing the rapid generation of genetically modified mice including the mutant mice at high efficiency and low mosaic rates. For in vitro fertilization, begin by super ovulating four or eight-week-old female C57BL/6J mice via intraperitoneal injection of 7.5 international units of pregnant mare serum gonadotropin followed by intraperitoneal injection of 7.5 international units of human chorionic gonadotropin 48 hours later.Next, remove cauda epididymis from sexually mature three to five-month-old male C57BL/6J mice and use a dissecting needle to extract clots of spermatozoa. Then incubate the clots in a 200 microliter drop of HTF at 37 degrees Celsius and 5%carbon dioxide for 1.5 hours for capacitation. 16 to 18 hours after human chorionic gonadotropin injection, collect cumulus oocyte complexes from the oviducts and incubate the complexes with 200 microliters of HTF covered with liquid paraffin for a no more than two-hour incubation in the cell culture incubator.At the end of the incubation, add one to five microliters of sperm suspension from the boundary of the incubation medium to the 200 microliter drop of cumulus oocyte complexes and place the co-culture in the cell culture incubator for three hours. At the end of the incubation, wash the oocytes with potassium supplemented simplex optimization medium three times to remove any remaining sperm and cumulus cells and use an inverted microscope to check the pronuclear formation and grade of the one cell embryos. For one cell embryo cryopreservation, transfer the embryos into a fresh 200 microliter drop of HTF supplemented with 20%fetal bovine serum for a 10-minute incubation in the cell culture incubator.At the end of the incubation, transfer 20 to 100 embryos to 50 microliters of one molar dimethyl sulfoxide solution and transfer up to 100 embryos in five microliters of solution to the bottom of a cryotube. Cool the embryos for five minutes at zero degrees Celsius before adding 45 microliters of DAP213 solution down the side of the tube. After capping, keep the cells for an additional five minutes at zero degrees Celsius before quickly storing the tube in liquid nitrogen.Prepare one microgram per microliter of CRISPR RNA and one microgram per microliter of tracrRNA in reduced serum minimal essential medium. Add six microliters of each RNA solution to 42 microliters of nuclease-free buffer and place the mixture into a dry heater for three minutes at 95 degrees Celsius. At the end of the incubation, cool the mixture at room temperature for five minutes and prepare one microgram per microliter of HiFi Cas9 protein with reduced serum minimal essential medium.Add six microliters of the diluted HiFi Cas9 solution to the RNA solution and transfer up to 100 thawed embryos into 25 microliters of the resulting ribonucleoprotein complex solution in a one whole glass slide. Then incubate embryos for 10 minutes at 37 degrees Celsius. For embryo electroporation, at the end of the incubation, set the electroporator parameters and load the slide onto the electroporator.Electroporation should be performed within one hour of embryo thawing to ensure that genome editing occurs before the first replication of DNA and to prevent mosaicism. After the electroporation, wash the embryos three times with modified Krebs-Ringer bicarbonate buffer two medium in two washes with a drop of potassium supplemented simplex optimization medium covered with liquid paraffin. Then incubate the embryos in fresh potassium supplemented simplex optimization medium in the cell culture incubator overnight.Using this modified cryopreservation method for one-cell embryos improves the developmental rate of the freeze-thawed embryos to the two-cell stage. Using this method, all of the generated mice in this representative experiment were albino with only one mosaic mouse bearing a coat with white and black patches. Here a representative section of a chromatogram from an albino mouse containing the M1 allele can be observed.As illustrated, the protocol can generate knockout and knock-in mice with high efficiency and low mosaic rates. Indeed, the F1 offspring of homozygous F0 offspring are heterozygous containing one mutant and one wild type allele with no disparate mutations. Remember that the fertilized egg is fragile at one-cell stage.The addition of fetal bovine serum reinforces the embryo. However, it must be handled carefully at each step. This method can contribute to the analysis of gene function and research in human diseases by facilitating a faster and more efficient production of genetically modified mice.

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