Name: mouse in vivo placental targeted crispr manipulation
Uploaded: 2023-04-14T14:40:00
Description: Watch this Scientific Journal Video about Mouse In Vivo Placental Targeted CRISPR Manipulation at JoVE.com

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>
<p class="jove_...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Future+horizons+for+neurodevelopmental+disorders:+Placental+mechanisms.">Future horizons for neurodevelopmental disorders: Placental mechanisms.</a> Frontiers in Pediatrics. 9, 653230 (2021).</li><li>Woods, L., Perez-Garcia, V., Hemberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+placental+development+and+its+impact+on+fetal+growth-new+insights+from+mouse+models.">Regulation of placental development and its impact on fetal growth-new insights from mouse models.</a> Frontiers in Endocrinology. 9, 570 (2018).</li><li>Goeden, N., 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=Maternal+Inflammation+disrupts+fetal+neurodevelopment+via+increased+placental+output+of+serotonin+to+the+fetal+brain.">Maternal Inflammation disrupts fetal neurodevelopment via increased placental output of serotonin to the fetal brain.</a> Journal of Neuroscience. 36 (22), 6041-6049 (2016).</li><li>vander Bom, T., 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=The+changing+epidemiology+of+congenital+heart+disease.">The changing epidemiology of congenital heart disease.</a> Nature Reviews Cardiology. 8 (1), 50-60 (2011).</li><li>Wilson, R. 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L., Leone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Cre+recombinase+in+early+diploid+trophoblast+cells+of+the+mouse+placenta.">Expression of Cre recombinase in early diploid trophoblast cells of the mouse placenta.</a> Genesis. 45 (3), 129-134 (2007).</li><li>Zhou, C. C., 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=Targeted+expression+of+Cre+recombinase+provokes+placental-specific+DNA+recombination+in+transgenic+mice.">Targeted expression of Cre recombinase provokes placental-specific DNA recombination in transgenic mice.</a> PLoS One. 7 (2), e29236 (2012).</li><li>Wattez, J. S., Qiao, L., Lee, S., Natale, D. R. 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N., Crombleholme, T., Habli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+placental+gene+transfer+of+IGF-1+corrects+placental+insufficiency+via+enhanced+placental+glucose+transport+mechanisms.">Adenoviral-mediated placental gene transfer of IGF-1 corrects placental insufficiency via enhanced placental glucose transport mechanisms.</a> PLoS One. 8 (9), e74632 (2013).</li><li>Jones, H., Crombleholme, T., Habli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+amino+acid+transporters+by+adenoviral-mediated+human+insulin-like+growth+factor-1+in+a+mouse+model+of+placental+insufficiency+in+vivo+and+the+human+trophoblast+line+BeWo+in+vitro.">Regulation of amino acid transporters by adenoviral-mediated human insulin-like growth factor-1 in a mouse model of placental insufficiency in vivo and the human trophoblast line BeWo in vitro.</a> Placenta. 35 (2), 132-138 (2014).</li><li>Song, A. 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K., Collen, D., VandenDriessche, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosafety+of+adenoviral+vectors.">Biosafety of adenoviral vectors.</a> Current Gene Therapy. 3 (6), 527-543 (2003).</li><li>Evers, 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=CRISPR+knockout+screening+outperforms+shRNA+and+CRISPRi+in+identifying+essential+genes.">CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes.</a> Nature Biotechnology. 34 (6), 631-633 (2016).</li><li>Rossant, J., Cross, J. 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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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+primary+mouse+trophoblast+cells+and+trophoblast+invasion+assay.">Isolation of primary mouse trophoblast cells and trophoblast invasion assay.</a> Journal of Visualized Experiments. (59), e3202 (2012).</li><li>Mandegar, M. 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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;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 &#956;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&#8482; 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 &#38; 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&#8482; 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 &#38; 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&#8482; Probe- Mm-Prl8a8-C2</td>
			<td >Advanced Cells Diagnostics</td>
			<td >&#160;528641-C2</td>
			<td></td>
		</tr>
		<tr >
			<td >RNAscope&#8482; 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&#185;/&#8322;</td>
		</tr>
		<tr >
			<td >Sodium Chloride (Saline)</td>
			<td >Hospra</td>
			<td >NDC 0409-4888-03</td>
			<td>Sterile,&#160; 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&#8482; Invitrogen&#8482; Nanodrop&#8482; One Spectrophotometer with WiFi and Qubit&#8482; 4 Fluorometer</td>
			<td >Fisher Scientific</td>
			<td >13-400-525</td>
			<td>This configuration comes with Qubit 4 fluorometer.&#160; 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&#8482; 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&#8482; 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.&#160; 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 ...

This protocol is significant as it allows for time-specific targeted manipulation of mouse placentas using CRISPR. This will allow researchers to elucidate specific gene functions during mid to late pregnancy. Genetic manipulations causing overexpression within the mouse placenta are very uncommon.The main advantage of this technique is it allows for a range of gene expression changes within the placenta. Do not get discouraged if preliminary terms have this technique are unsuccessful. As this technique is challenging, it requires quite a bit of time to practice to master.To begin, place the anesthetized dam supine with a nose cone for maintaining anesthesia in the preparation area. Shave the abdomen of the dam thoroughly. To prevent corneal drying, place artificial tear gel over both eyes of the dam.Then use sterile cotton tipped applicators to disinfect the surgical site with at least three alternating rounds of a povidone-iodine solution, followed by 70%ethanol with a final coat of povidone-iodine solution. After preparation, move the dam with the anesthesia nose cone to the designated surgery area. For the uterine laparotomy, place the dam supine on the operating table and secure the nose cone in place with tape.Place a heating pad underneath an absorbent pad set to 45 degrees Celsius. After confirming the anesthesia depth via lack of toe pinch reflux, use forceps and scissors to make an approximate two-centimeter midline incision through the tented up skin. Then make a vertical incision through the peritoneum using sterile forceps, gently tented away from the uterine horns.Massage both uterine horns by pressing on either side of the abdomen with fingers to guide them. Place the exposed uterus on top of sterile surgical drapes placed over the lower abdomen and keep it moist with drops of warm sterile saline as necessary. Once the uterus is exposed, select three pairs of placentas for manipulation.Record the location of placental manipulations and the organization of the embryos within the uterine horns. For the placental injection of the control plasmid, load a sufficient quantity of the appropriate control plasmid for three injections using a calibrated micropipette. Perform all the injections between the decidua and junctional zone at a depth of approximately 0.5 millimeters laterally into the placenta.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. Within two minutes of injection, perform electroporation using a pair of three-millimeter paddles attached to an electroporation machine. To ensure CRISPR incorporation efficiency and the viability of embryos, adjust the settings to 30 volts, 30-millisecond pulse, two pulses, 970-millisecond pulse off, and unipolar.Gently press the electroporation paddles over the uterine walls on the lateral sides of the placenta. Place the anode paddle over the injection site and the cathode directly opposite. Press the pulse on the electroporation machine and wait for the two pulses to complete prior to removing the electroporation paddles.Then proceed to placental injection of the experimental plasmid as demonstrated earlier. Perform electroporation of all three experimental injected placentas within two minutes of the injection as performed for the control group. Once the placental manipulation is complete, gently massage the uterine horns back inside the abdominal cavity using only fingers to directly manipulate the uterine horns.Perform interrupted sutures on the peritoneum layer using 45 centimeters long coated and braided dissolvable sutures with a 13-millimeter, three-eighths circle needle alloy. After suturing the peritoneum layer, suture the skin with dissolvable sutures using the same type of dissolvable sutures used for the peritoneum. Once the suturing is complete, apply tissue adhesive to the sutures on the skin.When the tissue adhesive has dried, turn off the vaporizer and transfer the dam from the surgery area to a supportive cage on its back. Placentas were injected with a general CRISPR Cas9 control plasmid and activation control CRISPR plasmid or an IGF-1 SAM activation plasmid. The representative post-necropsy images showed no noticeable damage or change in the phenotypic appearance in any treatment groups.The survival rate of the untreated embryos in manipulated litters decreased to an average of 79.05%while there was a significant decrease in the survival of the manipulated embryos averaging 55.56%There was no significant difference in any group's placental weight. There was no significant difference in maternal weight gain immediately following surgery compared to sham surgeries. Many pregnant dams displayed a slight decrease or no change in weight the day after the procedure, likely due to disrupted eating during and briefly after the surgery.Most dams showed increased weight on E14.5 but occasionally a decrease in weight was still observed. The qPCR showed a significant increase in IGF-1 expression in the IGF-1 overexpression placentas versus the control placentas. The ELIZA assessment of the E14.5 placentas showed a significant increase in IGF-1 protein levels in the IGF-1 overexpression placentas versus the control placentas.Green autofluorescence demonstrated the sub regions of the placental maternal fetal interface with red Cas9 labeling in only the IGF-1 overexpression placenta, indicating successful CRISPR incorporation in all three sub regions of the placenta. The fluorescent in C2 hybridization labeling confirmed the incorporation of the IGF-1 activation plasmid with no migration into untreated placentas. The decidual and labyrinth zone could be identified surrounding the red junctional zone marked by Prl8a8 staining.Remember to record the manipulation type and location of placentas and corresponding embryos. Recording the location of the cervix and embryos within the uterine horns is highly recommended. Following this procedure, analysis of the placenta, embryo, and dam can be performed.This will allow for a greater understanding of genes placental specific roles in pregnancy, placental function, and embryonic development.

mouse-in-vivo-placental-targeted-crispr-manipulation

Research

JoVE Journal

Genetics

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

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 ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

Manipulation of Gene Function in Mexican Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.&#160;

We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...