The authors acknowledge the following funding sources: R01 MH122435, NIH T32GM008629, and NIH T32GM145441. The authors thank Dr. Val Sheffield and Dr. Calvin Carter&#39;s labs at the University of Iowa for the use of their surgery room and equipment, as well Dr. Eric Van Otterloo, Dr. Nandakumar Narayanan, and Dr. Matthew Weber for their assistance with microscopy. The authors also thank Dr. Sara Maurer, Maya Evans, and Sreelekha Kundu for their assistance with the pilot surgeries.

All procedures were performed in accordance and compliance with federal regulations and University of Iowa policy and were approved by Institutional Animal Care and Use Committee.
1. Animals and husbandry
<ol>
	<li>Keep the animals in a 12 h daylight cycle with food and water ad libitum.</li>
	<li>Use CD-1 female mice aged 8-15 weeks. Use the presence of a copulatory plug to identify E0.5.</li>
	<li>On E0.5, singly house the pregnant dams.</li>
</ol>
2. Calibration of the micropipette
NOTE: The calibration of the micropipette should be performed prior to surgery when possible.
<ol>
	<li>Prior to making and calibrating the micropipette, dilute all the plasmids to the recommended concentration (0.1 &#181;g/&#181;L) in DNase-free water. Mix the plasmid with appropriately diluted Fast Green dye (1 &#181;g/&#181;L in PBS) (final plasmid concentration: 0.06 &#181;g/&#181;L).</li>
	<li>Pull micropipettes using 10 cm glass capillaries with an outer diameter of 1.5 mm and an inner diameter of 0.86 mm with a micropipette puller.</li>
	<li>Carefully break off the tip of a micropipette (2-3 mm) with sterile forceps.</li>
	<li>Load the micropipette into a microcapillary tip attached to a microinjector. Ensure the microinjector is attached to the microinjection machine and that there are sufficient levels of nitrogen to calibrate the micropipette.</li>
	<li>Once the micropipette is attached to the microinjector, load it with the Fast Green dye solution to perform the calibration (1 &#181;g/&#181;L in PBS). Calibrate the micropipette to inject a volume of approximately 3.5 &#181;L. Empty (&#34;clear&#34;) the micropipette in preparation for loading the plasmid solutions as below. 
	&#8203;NOTE: Each micropipette will be slightly different. To avoid damaging the placenta during the injection, the injection time should be set between 0.5-1.5 s. The pressure should be set between 1-8.5 psi. Calibrate each micropipette separately; if the micropipette cannot be calibrated within these parameters, then it should not be used.</li>
</ol>
3. Surgery (Figure 1A)
NOTE: To prepare, clean the surfaces of both the preparation and surgical areas with 70% ethanol. Place an absorbent underpad in the preparation area. In the surgery area, place a heating pad down, and then place an absorbent underpad on top of this. Sterilize all the tools prior to surgery. The time the dam is under anesthesia should be under 1 h.
<ol>
	<li>Anesthetization
	<ol>
		<li>Administer 5 mg/kg of NSAID (meloxicam) or another approved analgesic to the pregnant dam 30 min to 1 h before surgery.</li>
		<li>Place the pregnant dam in an induction chamber attached to an isoflurane vaporizer.</li>
		<li>Set the vaporizer to 4% isoflurane and 3.5 L/min oxygen.</li>
		<li>Once anesthetization has been confirmed by the lack of response to a toe pinch and a reduced breathing rate, remove the dam from the induction chamber to the preparation area.</li>
	</ol>
	</li>
	<li>Surgery preparation
	<ol>
		<li>In the preparation area, place the dam supine with a nose cone.</li>
		<li>Reduce the vaporizer to 2% isoflurane and 3.5 L/min oxygen while the dam is in the nose cone.</li>
		<li>Shave the abdomen of the dam thoroughly&#160;and remove excess fur. Alternate coating the shaved abdomen with povidone solution and 70% ethanol three times using sterile cotton-tipped applicators. Apply final coat of povidone solution. To prevent corneal drying, place artificial tear gel over both eyes of the dam (Figure 2A, B).</li>
		<li>After preparation, move the dam with the nose cone to the designated surgery area.</li>
	</ol>
	</li>
	<li>Uterine laparotomy 
	NOTE: Use sterile gloves throughout the surgical procedure. Change the gloves if any non-sterile surface is contacted.&#160;
	<ol>
		<li>In the surgery area, place the dam supine, and secure the nose cone in place with tape. Set the heating pad underneath the absorbent pad to 45 &#176;C.</li>
		<li>Using forceps and scissors, make an approximate 2 cm midline incision through the skin. Use forceps to tent the skin, and make a vertical incision into the skin. After this, make another similarly sized incision through the peritoneum to expose the uterine horns. Using forceps, tent the peritoneum while making the vertical incision (Figure 2C, D). 
		NOTE: Failure to properly tent while incising the peritoneum may lead to a fatal incision of the intestines.</li>
		<li>Gently massage the uterine horns through the incision by pressing on the sides of the abdomen. Do this by carefully guiding the uterus without tools, using only fingers to avoid accidental injury. Place the exposed uterus on top of a sterile surgical drape&#160;covering the abdomen of the dam&#160;and keep it moist throughout surgery with drops of sterile saline, which can be warmed to 30 &#176;C prior to use as needed (Figure 2E, F). 
		NOTE: The uterine horns can be identified as described in Wang et al.27.</li>
		<li>Once the uterus is exposed, select three pairs of placentas for manipulation. 
		NOTE: The placentas can be identified as described by Elmore et al.24. To maximize the survival of the embryos, no more than 6 placentas should be treated. If there are fewer than 12 embryos present, no more than 4 placentas should be injected. Select two adjacent placentas so that one receives a control injection and the other receives the experimental plasmid. The use of two adjacent placentas allows for a better comparison of placentas in similar locations in the uterus and also enables an increased rate of survival. The selected placental pairs are chosen randomly and spaced throughout both uterine horns (Figure 1B).</li>
		<li>Record the location of placental manipulations and organization of the embryos within the uterine horns so that the embryos and placentas can be identified during collection, as one dam will carry both control and experimentally treated placentas/embryos.</li>
	</ol>
	</li>
	<li>Placental injection and electroporation of the control plasmid 
	NOTE: To maintain an aseptic technique, sterilize the electroporation paddles and microinjector with a cold sterilant before use. Change gloves if any non-sterile surface is contacted.&#160;
	<ol>
		<li>Using the calibrated micropipette, load a sufficient quantity of the appropriate control plasmid for three injections. Perform all the injections at a depth of ~0.5 mm laterally into the placenta between the decidua (white) and junctional zone (dark red) (Figure 3A-F).</li>
		<li>Perform injections in the three control placentas. 
		NOTE: Perform all the control plasmid injections prior to the experimental injections to avoid cross-contamination of the plasmids with the micropipette. This will allow for the same micropipette to be used, as changing micropipettes and calibration time drastically increases the surgery time and decreases dam survival.</li>
		<li>Perform electroporation of the control plasmid-injected placentas within 2 min of the injection.</li>
		<li>For electroporation, use a pair of 3 mm paddles attached to an electroporation machine. To ensure CRISPR incorporation efficiency and the viability of embryos, use the following electroporation settings: 2 pulses, 30 V, 30 ms pulse, 970 ms pulse off, unipolar.</li>
		<li>After injection but immediately prior to electroporation, coat the places of contact with sterile saline, applying the saline precisely to the three sites on the uterine wall and the paddles with a dropper or syringe.</li>
		<li>Gently press the electroporation paddles on the lateral sides of the placenta. Place the anode paddle over the injection site and the cathode directly opposite (Figure 4A-C).</li>
		<li>Press the pulse on the electroporation machine, and wait for the two pulses to complete prior to removing the electroporation paddles. 
		NOTE: A small amount of white foam is often seen between the paddles and placenta during pulses. If this does not occur, check the voltage from the paddles with a voltmeter before further use. If the reading on the voltmeter does not match the electroporation voltage setting, the paddles are nonfunctional.</li>
	</ol>
	</li>
	<li>Placental injection and electroporation of the experimental plasmid
	<ol>
		<li>Follow the same instructions from step 3.4.1. to step 3.4.2. using the experimental plasmid instead of the control plasmid.</li>
		<li>Perform electroporation of all three experimental injected placentas within 2 min of the injection. This should be performed in the same manner as for those injected with the control plasmids. Follow step 3.4.4. to step 3.4.7.</li>
	</ol>
	</li>
	<li>Completion of the surgery
	<ol>
		<li>Once the placental manipulation is complete, gently massage the uterine horns back inside the abdominal cavity using only fingers (Figure 5A).</li>
		<li>First, perform double-knotted single sutures on the peritoneum layer using coated and braided dissolvable sutures that are 45 cm long with a 13 mm 3/8c needle alloy. Space the sutures 2-3 mm apart (Figure 5B).</li>
		<li>After suturing the peritoneum layer, suture the skin with dissolvable sutures. Triple knot the single sutures 2-3 mm apart to ensure that the dam cannot undo the suturing (Figure 5C).</li>
		<li>Once the suturing is complete, set the isoflurane to 1% and the oxygen to 3.5 L/min, and apply tissue adhesive to the sutures on the skin (Figure 5D). 
		NOTE: Tissue adhesive is optional but recommended to prevent re-opening of the incision due to dam chewing.</li>
		<li>When the tissue adhesive has dried, turn off the vaporizer, and remove the dam from the surgery area. Place the dam in a supportive cage on its back.</li>
	</ol>
	</li>
</ol>
4. Post-surgery care and monitoring
<ol>
	<li>Allow the pregnant dam to recuperate in a clean cage under supervision for a minimum of 30 min to ensure no immediate complications of the surgery. Observe until it is fully ambulatory and can flip onto its feet without assistance. Singly house the dam post-surgery.</li>
	<li>Follow the institutional post-operative care and monitoring until embryo collection. Record the dam weight, and monitor the sutures and the incision site daily.</li>
</ol>
5. E14.5 placental collection
<ol>
	<li>On E14.5, deeply anesthetize the dam with a ketamine/xylazine cocktail (1 mg/mL ketamine and 0.1 mg/mL xylazine), and then cervically dislocate the dam.</li>
	<li>Make a V-shaped incision into the abdomen of the dam with scissors, and remove the uterus. Place immediately onto a 5 cm Petri dish on ice. Using forceps, carefully remove the embryo and the corresponding placenta from the uterus. 
	NOTE: Keep a record of the embryo location and corresponding placenta in the uterine horns to determine which received the direct manipulation during surgery.</li>
	<li>Record the placental weights. Using RNAse-free forceps and razors, cut the placenta in half down the midline. Put one half into 4% PFA at 4 &#176;C. Cut the remaining half in half again, and store the remaining two quarters at &#8722;80 &#176;C in two tubes, one with RNA storage reagent. 
	&#8203;NOTE: Embryos and other maternal tissues can be stored from the collection at &#8722;80 &#176;C for future use.</li>
</ol>
6. Placental gene expression analysis
<ol>
	<li>Use the quarter of the placenta stored at &#8722;80 &#176;C in RNA storage reagent.</li>
	<li>Process the placentas for qPCR as described in Elser et al. using the Trizol method for RNA isolation, a spectrophotometer for RNA concentration, a cDNA synthesis kit, and qPCR with SYBR Green Master Mix28. In place of the Turbo DNAfree kit DNAse referenced in Elser et al., use an RNAcleanup kit after the Trizol RNA isolation to ensure the samples do not contain contaminants28. 
	NOTE: Read the material safety data sheet (MSDS) for Trizol, and use it in a chemical fume hood.</li>
	<li>Assess the plasmid insertion of the control plasmid with GFP primers and of the experimental plasmid with BLAST primers (primers listed in Supplementary Table 1). Use the CT value to determine the presence of the plasmid. 
	NOTE: CT values above 35 are false positives, and only those below 35 should be considered a positive indicator that the plasmid was successfully inserted.</li>
	<li>Assess IGF-1 placental expression normalized to the housekeeping gene 18s (primers listed in Supplementary Table 1). Use the ddCT method to calculate the fold change, and then calculate the normalized fold change of the experimental samples to the mean fold change of the control samples.</li>
</ol>
7. Placental protein level analysis
<ol>
	<li>Use the quarter of the placenta that has been stored at &#8722;80 &#176;C. Homogenize the tissue in a buffer solution made of 11.5 mM Tris HCl, 5 mM MgCl2, and 10 mM protease inhibitor in diH2O with a final pH of 7.4. Use a handheld homogenizer and pestle to break up the tissue. 
	NOTE: The sample should not exceed 10% of the volume of the buffer.</li>
	<li>Dilute the homogenized samples in the buffer at 1:12, so that they are within the detectable range of a bicinchoninic acid assay (BCA) kit. Perform the BCA assay according to the manufacturer&#39;s instructions, and quantify the total protein using a plate reader.</li>
	<li>After performing the BCA assay, normalize all the samples to the same total protein concentration of 2 mg/mL for the IGF-1 ELISA, as done in Gumusoglu et al.29.</li>
	<li>Perform the IGF-1 ELISA according to the manufacturer&#39;s instructions, and quantify the IGF-1 protein levels with a plate reader using a standard concentration curve.</li>
</ol>
8. Spatial CRIPSR verification using fluorescent in situ hybridization labeling
<ol>
	<li>After the placental halves have been appropriately fixed in 4% PFA at 4 &#176;C for 1-3 days, move them into 20% sucrose at 4 &#176;C before freezing them in optimal cutting temperature (OCT) compound.</li>
	<li>Serially section the OCT-embedded placenta halves in a &#8722;20 &#176;C cryostat into 10 &#181;m sections, and place them onto slides to be labeled. Section the placenta halves so that all three subregions are visible. Store the slides at &#8722;80 &#176;C until use for in situ hybridization.</li>
	<li>Perform fluorescence in situ hybridization (FISH) labeling following the manufacturer&#39;s protocols. Hybridize one slide with a dCas9-3xNLS-VP64 probe and a second &#34;sister&#34; slide of the same placenta with a Prl8a8 probe. 
	NOTE: The dCas9-3xNLS-VP64 probe detects the presence of the overexpression plasmid. The Prl8a8 probe highlights spongiotrophoblasts of the junctional zone, which allows for the subregions of the placenta to be identifiable. These two probes are labeled on separate &#34;sister&#34; slides to avoid interference of multi-channel fluorescence with the green autofluorescence in the placentas.</li>
	<li>Label both probes with the detection dye (Opal 620). After completing the manufacturer&#39;s protocol for FISH, apply DAPI mounting medium, and place a coverslip on the slide. Seal the coverslip with clear nail polish.</li>
	<li>Image the slides on an upright compound fluorescence microscope, and process them using an appropriate image processing software. Here, the CellSens software was used.</li>
</ol>

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+maternal+interleukin-17+and+autism-related+cortical+gene+expression,+neurobiology,+and+behavior.">Chronic maternal interleukin-17 and autism-related cortical gene expression, neurobiology, and behavior.</a> Neuropsychopharmacology. 45 (6), 1008-1017 (2020).</li><li>Liu, F., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+gene+transfer+to+the+liver+following+systemic+administration+of+plasmid+DNA.">Electric gene transfer to the liver following systemic administration of plasmid DNA.</a> Gene Therapy. 9 (16), 1116-1119 (2002).</li><li>Kalli, C., Teoh, W. C., Leen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+genes+via+sonoporation+and+electroporation.">Introduction of genes via sonoporation and electroporation.</a> Advances in Experimental Medicine and Biology. 818, 231-254 (2014).</li><li>Wu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+vivo+gene+editing+using+ribonucleoproteins+in+skin+stem+cells+of+recessive+dystrophic+epidermolysis+bullosa+mouse+model.">Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (7), 1660-1665 (2017).</li><li>Nakamura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electroporation and Sonoporation in Developmental Biology. , (2009).</li><li>Bond, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+timing+and+coordination+of+neurogenesis+and+astrogenesis+in+developing+mouse+hippocampal+subregions.">Differential timing and coordination of neurogenesis and astrogenesis in developing mouse hippocampal subregions.</a> Brain Sciences. 10 (12), 909 (2020).</li><li>Kojima, Y., Tam, O. H., Tam, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+developmental+events+in+the+early+mouse+embryo.">Timing of developmental events in the early mouse embryo.</a> Seminars in Cell &amp; Developmental Biology. 34, 65-75 (2014).</li><li>Pennington, K. A., Schlitt, J. M., Schulz, L. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+Interference+efficiently+induces+specific+and+reversible+gene+silencing+in+human+iPSCs.">CRISPR Interference efficiently induces specific and reversible gene silencing in human iPSCs.</a> Cell Stem Cell. 18 (4), 541-553 (2016).</li><li>Dai, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+CRISPRa+screen+identifies+putative+enhancers.">Inducible CRISPRa screen identifies putative enhancers.</a> Journal of Genetics and Genomics. 48 (10), 917-927 (2021).</li><li>Ursini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+genomic+risk+scores+and+early+neurodevelopmental+outcomes.">Placental genomic risk scores and early neurodevelopmental outcomes.</a> Proceedings of the National Academy of Sciences of the United States of America. (7), e2019789118 (2021).</li><li>Smajdor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethical+challenges+in+fetal+surgery.">Ethical challenges in fetal surgery.</a> Journal of Medical Ethics. 37 (2), 88-91 (2011).</li><li>Antiel, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethical+challenges+in+the+new+world+of+maternal-fetal+surgery.">Ethical challenges in the new world of maternal-fetal surgery.</a> Seminars in Perinatology. 40 (4), 227-233 (2016).</li></ol>

The authors have nothing to disclose.

The placenta is a primary regulator of fetal growth, and as previously noted, changes in placental gene expression or function may significantly impact fetal development6. The protocol outlined here can be used to perform a targeted in vivo CRISPR manipulation of the mouse placenta using a relatively advanced surgical approach. This technique allows for a significant yield of viable embryos and their corresponding placentas that can be used for further study (Figure 6...

The placenta is an essential organ involved in the development of the fetus. The main role of the placenta is to provide essential factors and regulate the transfer of nutrients and waste to and from the fetus. Mammalian placentas are composed of both fetal and maternal tissue, which make up the fetal-maternal interface, and, thus, the genetics of both the mother and fetus impact function1. Genetic anomalies or impaired function of the placenta can drastically alter fetal development. Previous work has shown that placental genetics and development are associated with the altered development of specific organ systems in the fetus. Particularly, abnormalities in the placenta are linked with changes in the fetal brain, heart, and vascular system2,3,4,5.
The transport of hormones, growth factors, and other molecules from the placenta to the fetus plays a major role in fetal development6. It has been shown that altering the placental production of specific molecules can alter neurodevelopment. Maternal inflammation can increase the production of serotonin by altering tryptophan (TRP) metabolic gene expression in the placenta, which subsequently creates an accumulation of serotonin in the fetal brain7. Other studies have found placental abnormalities alongside heart defects. Abnormalities in the placenta are thought to contribute to congenital heart defects, the most common birth defect in humans8. A recent study has identified several genes that have similar cellular pathways in both the placenta and heart. If disrupted, these pathways could cause defects in both organs9. The defects in the placenta may exacerbate congenital heart defects. The role of placental genetics and function on specific fetal organ system development is an emerging field of study.
Mice have hemochorial placentas and other features of human placentas, which makes them highly useful models for studying human disease1. Despite the importance of the placenta, there is currently a lack of targeted in vivo genetic manipulations. Furthermore, there are currently more options available for knockouts or knockdowns than overexpression or gain-of-function manipulations in the placenta10. There are several transgenic Cre-expressing lines for placental-specific manipulation, each in different trophoblast lineages at different time points. These include Cyp19-Cre, Ada/Tpbpa-Cre, PDGFR&#945;-CreER, and Gcm1-Cre11,12,13,14. While these Cre transgenes are efficient, they may not be capable of manipulating some genes at specific time points. Another commonly used method to either knockout or overexpress placental gene expression is the insertion of lentiviral vectors into blastocyst culture, which causes a trophoblast-specific genetic manipulation15,16. This technique allows for a robust change in the placental gene expression early in development. The use of RNA interference in vivo has been sparsely utilized in the placenta. The insertion of shRNA plasmids can be performed similarly to the CRIPSR technique described in this paper. This has been done at E13.5 to successfully decrease PlGF expression in the placenta, with impacts on offspring brain vasculature17.
In addition to techniques that are primarily used for knockout or knockdown, inducing overexpression is commonly performed with adenoviruses or the insertion of an exogenous protein. The techniques used for overexpression have varying rates of success and have mostly been performed later in gestation. To investigate the role of insulin-like growth factor 1 (IGF-1) in placental function, an adenoviral-mediated placental gene transfer was performed to induce the overexpression of the IGF-1 gene18,19. This was performed late in mouse gestation on E18.5 via&#160;direct placental injection. To provide additional options and circumvent possible failures of established placental genetic manipulations, such as Cre-Lox combination failures, the possible toxicity of adenoviruses, and the off-target effects of shRNA, in vivo direct CRISPR manipulation of the placenta can be used20,21,22. This model was developed to address the lack of overexpression models and to create a model with flexibility.
This technique is based upon the work of Lecuyer et al., in which shRNA and CRISPR plasmids were targeted directly in vivo to mouse placentas to alter PlGF expression17. This technique can be used to directly alter placental gene expression using CRISPR manipulation at multiple time points; for this work, E12.5 was selected. The placenta has matured by this point and is large enough to manipulate, allowing for the insertion of a specific CRISPR plasmid on E12.5, which can have a significant impact on fetal development from mid to late pregnancy23,24. Unlike transgenic approaches, but similar to viral inductions or RNA interference, this technique allows for overexpression or knockout at particular time points using a relatively advanced surgical approach, thus avoiding possible impaired placentation or embryonic lethality from earlier changes. As only a few placentas receive the experimental or control plasmid within a litter, the approach allows for two types of internal controls. These controls are those injected and electroporated with the appropriate control plasmid and those that receive no direct manipulation. This technique was optimized to create an overexpression of the IGF-1 gene in the mouse placenta via a synergistic activation mediator (SAM) CRISPR plasmid. The IGF-1 gene was chosen, as IGF-1 is an essential growth hormone delivered to the fetus that is primarily produced in the placenta prior to birth25,26. This new placental-targeted CRISPR technique will allow for direct manipulation to help define the connection between placental function and fetal development.

<table><tbody><tr><td>1.5 ml Tubes</td><td>USA Scientific Inc</td><td>1615-5500</td><td></td></tr><tr><td>4% Paraformeldhyde (PFA) in PBS</td><td>Thermo Fisher Scientific</td><td>J61899.AP</td><td></td></tr><tr><td>96 Well plate</td><td>Cornings</td><td>3598</td><td>For BCA kit</td></tr><tr><td>Absorbent Underpads</td><td>Fisher Scientific</td><td>14-206-62</td><td></td></tr><tr><td>Activation Control Plasmid</td><td>Santa Cruz Biotechnology</td><td>sc-437275</td><td>Dnase-free water provided for dilution</td></tr><tr><td>AMV Reverse Transcriptase</td><td>New England Biolabs</td><td>M0277L</td><td>Use for cDNA synthesis</td></tr><tr><td>Anesthetic Gas Vaporizor</td><td>Vetamac</td><td>VAD-601TT</td><td>VAD-compact vaporizer</td></tr><tr><td>Artifical Tear Gel</td><td>Akorn</td><td>NDC 59399-162-35</td><td></td></tr><tr><td>BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>23227</td><td>Protein quantification</td></tr><tr><td>Biovortexer</td><td>Bellco Glass, Inc.</td><td>198050000</td><td>Hand-held tissue homogenizer</td></tr><tr><td>CellSens Software</td><td>Olympus</td><td>V4.1.1</td><td>Image processing to FISH images.</td></tr><tr><td>Centrifuge 5810</td><td>Eppendorf</td><td>EP022628168</td><td>Plate centrifuge</td></tr><tr><td>Chloroform</td><td>Thermo Fisher Scientific</td><td>J67241-AP</td><td>RNA isolation</td></tr><tr><td>Cotton Tipped Applicators</td><td>ProAdvantage</td><td>77100</td><td>Sterilize before use</td></tr><tr><td>CRISPR/Cas9 Control Plasmid</td><td>Santa Cruz Biotechnology</td><td>sc-418922</td><td>Dnase-free water provided for dilution</td></tr><tr><td>CryoStat</td><td>Leica</td><td>CM1950</td><td></td></tr><tr><td>Dissection Microscope</td><td>Leica</td><td>M125 C</td><td>Used for post-necroscopy imaging</td></tr><tr><td>Dissolvable Sutures</td><td>Med Vet International</td><td>J385H</td><td></td></tr><tr><td>Distilled Water</td><td>Gibco</td><td>15230162</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS)</td><td>Thermo fisher Scientific</td><td>14190144</td><td>(-) Calcium; (-) Magnesium</td></tr><tr><td>ECM 830 Electro Electroporator (Electroporation Machine)</td><td>BTX Harvard Apparatus</td><td>45-0662</td><td>Generator only</td></tr><tr><td>Electric Razor</td><td>Wahl</td><td>CL9990</td><td>Kent Scientific</td></tr><tr><td>Electroporation paddles/Tweezertrodes</td><td>BTX Harvard Apparatus</td><td>45-0487</td><td>3 mm diameter paddles; wires included</td></tr><tr><td>Embedding Cassette: 250 PK</td><td>Grainger</td><td>21RK94</td><td>Placenta embedding cassettes</td></tr><tr><td>Ethanol</td><td>Thermo Fisher Scientific</td><td>268280010</td><td></td></tr><tr><td>F-Air Canisters</td><td>Penn Veterinary Supply Inc</td><td>BIC80120</td><td>Excess isoflurane filter</td></tr><tr><td>Fast Green Dye FCF</td><td>Sigma</td><td>F7252-5G</td><td>Dissolve to 1 &amp;mu;g/ml and filter; protect from light</td></tr><tr><td>Filter-based microplate photometer (plate reader)</td><td>Fisher Scientific</td><td>14377576</td><td>Can be used for BCA and ELISA</td></tr><tr><td>Forceps</td><td>VWR</td><td>82027-386</td><td>Fine tips, straight, serrated</td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Glass Capillaries - Borosilicate Glass (Micropipette)</td><td>Sutter Instrument</td><td>B150-86-10</td><td>O.D.: 1.5 mm, I.D.: 0.86 mm, 10 cm length</td></tr><tr><td>Halt Protease and Phosphotase inhibitor cocktail (100x)</td><td>Thermo Scientific</td><td>1861281</td><td>Protein homogenization buffer</td></tr><tr><td>Heating Pad</td><td>Thermotech</td><td>S766D</td><td>Digitial Moist Heating Pad</td></tr><tr><td>Hemostats</td><td>VWR</td><td>10806-188</td><td>Fully surrated jaw; curved</td></tr><tr><td>Hot Water Bath</td><td>Fisher Scientific</td><td>20253</td><td>Isotemp 205</td></tr><tr><td>Igf-1 SAM Plasmid (m1)</td><td>Santa Cruz Biotechnology</td><td>sc-421056-ACT</td><td>Dnase-free water provided for dilution</td></tr><tr><td>Induction Chamber</td><td>Vetamac</td><td>941443</td><td>No specific liter size required</td></tr><tr><td>Isoflurane</td><td>Piramal Pharma Limited</td><td>NDC 66794-013-25</td><td></td></tr><tr><td>Isoproponal/2-Proponal</td><td>Fisher Scientific</td><td>A451-4</td><td>RNA isolation</td></tr><tr><td>Ketamine HCl 100mg/ml</td><td>Akorn</td><td>NDC 59399-114-10</td><td></td></tr><tr><td>MgCl2/Magneisum Chloride</td><td>Sigma Aldrich</td><td>63069-100ML</td><td>1M. Protein homogenization buffer</td></tr><tr><td>MicroAmp&amp;trade; Optical 384-Well Reaction Plate with Barcode</td><td>Fisher Scientific</td><td>4309849</td><td>Barcoded plates not required</td></tr><tr><td>Microcapillary Tip</td><td>Eppendorf</td><td>5196082001</td><td>Attached to BTX Microinjector</td></tr><tr><td>Microinjector</td><td>BTX Harvard Apparatus</td><td>45-0766</td><td>Stainless Steel Pipette Holder, 130 mm Length, for 1 to 1.5 mm Pipettes</td></tr><tr><td>Microject 1000A (Injection Machine)</td><td>BTX Harvard Apparatus</td><td>45-0751</td><td>MicroJect 1000A Plus System</td></tr><tr><td>Micropipette Puller Model P-97</td><td>Sutter Instrument</td><td>P-97</td><td>Flaming/Brown type micropipette puller</td></tr><tr><td>Microplate Mixer (Plate Shaker)</td><td>scilogex</td><td>822000049999</td><td></td></tr><tr><td>Mouse/Rat IGF-I/IGF-1 Quantikine ELISA Kit</td><td>R &amp;amp; D Systems</td><td>MG100</td><td></td></tr><tr><td>Needles</td><td>BD - Becton, Dickson, and Company</td><td>305106</td><td>30 Gx 1/2 (0.3 mm x 13 mm)</td></tr><tr><td>Nitrogen Tank</td><td>Linde</td><td>7727-37-9</td><td>Any innert gas</td></tr><tr><td>Non-Steroidal Anti-Inflammatory Drug (NSAID)</td><td>Norbrook Laboratories Limited</td><td>NDC 55529-040-10</td><td>Analesgic such as Meloxicam</td></tr><tr><td>Nose Cone</td><td>Vetamac</td><td>921609</td><td>9-14 mm</td></tr><tr><td>Opal 620 detection dye</td><td>Akoya Biosciences</td><td>SKU FP1495001KT</td><td>Used for FISH</td></tr><tr><td>Optimal Cutting Temperature (O.C.T) Compound</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>Oxygen Tank</td><td>Linde</td><td>7782 - 44 - 7</td><td>Medical grade oxygen</td></tr><tr><td>Pestles</td><td>USA Scientific Inc</td><td>14155390</td><td></td></tr><tr><td>Povidone-Iodine Solution, 5%</td><td>Avrio Health L.P.</td><td>NDC 67618-155-16</td><td></td></tr><tr><td>Power SYBR&amp;trade; Green PCR Master Mix</td><td>Thermo Fisher Scientific</td><td>4367659</td><td>Use for qPCR</td></tr><tr><td>Random Hexamers (Random Primers)</td><td>New England Biolabs</td><td>S1330S</td><td>Use for cDNA synthesis</td></tr><tr><td>Razor Blade</td><td>Grainger</td><td>26X080</td><td></td></tr><tr><td>RNA Cleanup Kit &amp;amp; Concentrator</td><td>Zymo Research</td><td>R1013</td><td></td></tr><tr><td>RNALater</td><td>Thermo Fisher Scientific</td><td>AM7021</td><td></td></tr><tr><td>RNAscope kit v.2.5</td><td>Advanced Cells Diagnostics</td><td>323100</td><td>Contains all reagents required for fluorescent in situ hybridization. Probes sold separately.</td></tr><tr><td>RNAscope&amp;trade; Probe- Mm-Prl8a8-C2</td><td>Advanced Cells Diagnostics</td><td>&amp;nbsp;528641-C2</td><td></td></tr><tr><td>RNAscope&amp;trade; Probe- Vector-dCas9-3xNLS-VP64</td><td>Advanced Cells Diagnostics</td><td>527421</td><td></td></tr><tr><td>Roto-Therm Mini</td><td>Benchmark</td><td>R2020</td><td>Dry oven for in situ hybridization</td></tr><tr><td>Scissors</td><td>VWR</td><td>82027-578</td><td>Dissecting Scissors, Sharp Tip, 4&amp;sup1;/?</td></tr><tr><td>Sodium Chloride (Saline)</td><td>Hospra</td><td>NDC 0409-4888-03</td><td>Sterile,&amp;nbsp; 0.9%</td></tr><tr><td>Sodium Citrate, Trisodium Salt, Dihydrate, [Citric Acid, Trisodium Dihydrate]</td><td>Research Product International</td><td>03-04-6132</td><td></td></tr><tr><td>Sodium Hydroxide 1N Concentrate, Fisher Chemical</td><td>Fisher Scientific</td><td>SS277</td><td>Protein homogenization buffer</td></tr><tr><td>Steamer</td><td>Bella</td><td>B00DPX8UBA</td><td></td></tr><tr><td>Sterile Surgical Drape</td><td>Busse</td><td>696</td><td>Sterilize before use</td></tr><tr><td>Superfrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Surgipath Cover Glass 24x60</td><td>Leica</td><td>3800160</td><td></td></tr><tr><td>Syringes</td><td>BD - Becton, Dickson, and Company</td><td>309659</td><td>BD Luer Slip Tip Syringe sterile, single use, 1 mL</td></tr><tr><td>Thermo Scientific&amp;trade; Invitrogen&amp;trade; Nanodrop&amp;trade; One Spectrophotometer with WiFi and Qubit&amp;trade; 4 Fluorometer</td><td>Fisher Scientific</td><td>13-400-525</td><td>This configuration comes with Qubit 4 fluorometer.&amp;nbsp; Qubit quantification not required.</td></tr><tr><td>Tissue Adhesive</td><td>3M</td><td>1469SB</td><td>VetBond</td></tr><tr><td>Tris HCl</td><td>Thermo Fisher Scientific</td><td>15568025</td><td>1M. Protein homogenization buffer</td></tr><tr><td>TRIzol&amp;trade; Reagent</td><td>Thermo Fisher Scientific</td><td>15596018</td><td>RNA isolation</td></tr><tr><td>TSA Buffer Pack</td><td>Advanced Cells Diagnostics</td><td>322810</td><td>Used to dilute Opal 620 detection dye</td></tr><tr><td>Universal F-Circuit</td><td>Vetamac</td><td>40200</td><td>Attached to vaporizer and vaporizer accessories</td></tr><tr><td>Upright Compound Fluorescence Microscope</td><td>Olympus</td><td>BX61VS</td><td>Used for FISH imaging</td></tr><tr><td>Vectorshield with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td>Coverslip mounting media</td></tr><tr><td>ViiA&amp;trade; 7 Real-Time PCR System with 384-Well Block</td><td>Thermo Fisher Scientific</td><td>4453536</td><td>This is for SYBR 384-well block detection.&amp;nbsp; TaqMan and/or smaller blocks available</td></tr><tr><td>Wet n Wild Nail Polish Wild Shine, Clear Nail Protector, Nail Color</td><td>Amazon</td><td>C450B</td><td></td></tr><tr><td>Xylazine 20mg/ml</td><td>Anased</td><td>343730_RX</td><td></td></tr></tbody></table>

mouse in vivo placental targeted crispr manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

General procedure outcomes (Figure 6) 
In the study, there were three manipulated groups. These included placentas injected with a general CRISPR Cas9 control plasmid (Cas9 Control), an activation control CRISPR plasmid (Act Control), or an IGF-1 SAM activation plasmid (Igf1-OE). The Cas9 Control is better suited for knockout plasmids, and the activation control is better suited for overexpression/activation plasmids. To assess the viability ch...

Watch this Scientific Journal Video about Mouse In Vivo Placental Targeted CRISPR Manipulation at JoVE.com

Mouse In Vivo Placental Targeted CRISPR Manipulation

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

Mouse In Vivo Placental Targeted CRISPR Manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients ...

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2.4K Views.  University of Iowa. Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

Video: Mouse In Vivo Placental Targeted CRISPR Manipulation

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

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 ...

Developmental Biology

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. ...

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to assess therapeutic avenues. However, their time-, labor- and cost-intensive nature limits their utility for systematic analysis of gene function. Recent advances in genome-editing technologies overcome those limitations and allow for the rapid generation of specific gene perturbations directly within specific mouse organs in a multiplexed and rapid manner. Here, we describe a CRISPR/Cas9-based method (Clustered Regularly Interspaced Short Palindromic Repeats) to generate thousands of gene knock-out clones within the epithelium of the skin and oral cavity of mice, and provide a protocol detailing the steps necessary to perform a direct&#160;in vivo&#160;CRISPR&#160;screen for tumor suppressor genes. This approach can be applied to other organs or other CRISPR/Cas9 technologies such as CRISPR-activation or CRISPR-inactivation to study the biological function of genes during tissue homeostasis or in various disease settings.

Here we describe a rapid and direct in vivo CRISPR/Cas9 screening methodology using ultrasound-guided in utero embryonic lentiviral injections to simultaneously assess functions of several genes in the skin and oral cavity of immunocompetent mice.

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

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 ...

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.

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 ...

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 ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Bioengineering

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

Mouse In Vivo Placental Targeted CRISPR Manipulation

Summary

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Chapters in this video

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

Generation of Genetically Modified Mice through the Microinjection of Oocytes

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

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

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

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

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation