Name: crispr/cas9-mediated targeted integration in vivo using a homology-mediated end joining-based strategy
Uploaded: 2018-03-12T14:00:00
Description: Watch this Scientific Journal Video about CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy at JoVE.com

Questo lavoro è stato supportato da CAS strategico priorità Research Program (XDB02050007, XDA01010409), la nazionale Hightech di R & D programma (programma 863; 2015AA020307), National Natural Science Foundation of China (NSFC concede 31522037, 31500825, 31571509, 31522038), China Youth mille talenti programma (HY), rottura attraverso il progetto dell'Accademia delle scienze cinese, Shanghai città comitato della scienza e della tecnologia del progetto (16JC1420202 a HY), il Ministero della scienza e della tecnologia della Cina (la maggior parte; 2016YFA0100500).

Tutte le procedure tra cui animali soggetti sono state approvate dal comitato etico di ricerca biomedica presso gli istituti di Shanghai per scienze biologiche (CAS).
1. progettazione dei plasmidi di donatore
<ol><li>
Selezione di sgRNA
	<ol>
<li>Utilizzare strumenti di progettazione online CRISPR per prevedere sgRNAs la destinazione regione11,12,13,14</...

<ol>
	<li>Yang, H., 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=Generation+of+Mice+Carrying+Reporter+and+Conditional+Alleles+by+CRISPR/Cas-Mediated+Genome+Engineering.">Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering.</a> Cell. 154 (6), 1370-1379 (2013).</li><li>Hockemeyer, D., 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=Genetic+engineering+of+human+pluripotent+cells+using+TALE+nucleases.">Genetic engineering of human pluripotent cells using TALE nucleases.</a> Nature Biotechnology. 29 (8), 731-734 (2011).</li><li>Nakade, S., 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=Microhomology-mediated+end-joining-dependent+integration+of+donor+DNA+in+cells+and+animals+using+TALENs+and+CRISPR/Cas9.">Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9.</a> Nature Communications. 5, 5560 (2014).</li><li>Hisano, Y., 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=Precise+in-frame+integration+of+exogenous+DNA+mediated+by+CRISPR/Cas9+system+in+zebrafish.">Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish.</a> Scientific reports. 5, 8841 (2015).</li><li>Yao, X., 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=Cas9+-+Mediated+Precise+Targeted+Integration+In+Vivo+Using+a+Double+Cut+Donor+with+Short+Homology+Arms.">Cas9 - Mediated Precise Targeted Integration In Vivo Using a Double Cut Donor with Short Homology Arms.</a> EBioMedicine. , (2017).</li><li>Auer, T. O., Duroure, K., De Cian, A., Concordet, J. P., Del Bene, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+CRISPR/Cas9-mediated+knock-in+in+zebrafish+by+homology-independent+DNA+repair.">Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.</a> Genome research. 24 (1), 142-153 (2014).</li><li>Maresca, M., Lin, V. G., Guo, N., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obligate+ligation-gated+recombination+(ObLiGaRe):+custom-designed+nuclease-mediated+targeted+integration+through+nonhomologous+end+joining.">Obligate ligation-gated recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining.</a> Genome Research. 23 (3), 539-546 (2013).</li><li>Suzuki, K., 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=In+vivo+genome+editing+via+CRISPR/Cas9+mediated+homology-independent+targeted+integration.">In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration.</a> Nature. 540 (7631), 144-149 (2016).</li><li>Cong, L., 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=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Yao, X., 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=Homology-mediated+end+joining-based+targeted+integration+using+CRISPR/Cas9.">Homology-mediated end joining-based targeted integration using CRISPR/Cas9.</a> Cell Research. 27 (6), 801-814 (2017).</li><li>Han, D. 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=Direct+reprogramming+of+fibroblasts+into+epiblast+stem+cells.">Direct reprogramming of fibroblasts into epiblast stem cells.</a> Nature Cell Biology. 13 (1), 66-71 (2011).</li><li>Han, D. 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=Direct+Reprogramming+of+Fibroblasts+into+Neural+Stem+Cells+by+Defined+Factors.">Direct Reprogramming of Fibroblasts into Neural Stem Cells by Defined Factors.</a> Cell Stem Cell. , (2012).</li><li>Ambasudhan, R., 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=Direct+reprogramming+of+adult+human+fibroblasts+to+functional+neurons+under+defined+conditions.">Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions.</a> Cell Stem Cell. 9 (2), 113-118 (2011).</li><li>Sparman, 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=Epigenetic+reprogramming+by+somatic+cell+nuclear+transfer+in+primates.">Epigenetic reprogramming by somatic cell nuclear transfer in primates.</a> Stem Cells. 27 (6), 1255-1264 (2009).</li><li>Schatten, G., Mitalipov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+biology:+Transgenic+primate+offspring.">Developmental biology: Transgenic primate offspring.</a> Nature. 459 (7246), 515-516 (2009).</li><li>Hsu, P. D., 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=DNA+targeting+specificity+of+RNA-guided+Cas9+nucleases.">DNA targeting specificity of RNA-guided Cas9 nucleases.</a> Nature Biotechnology. 31 (9), 827-832 (2013).</li><li>Cong, L., 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=Multiplex+Genome+Engineering+Using+CRISPR/Cas+Systems.">Multiplex Genome Engineering Using CRISPR/Cas Systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Quadros, R. 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=Easi-CRISPR:+a+robust+method+for+one-step+generation+of+mice+carrying+conditional+and+insertion+alleles+using+long+ssDNA+donors+and+CRISPR+ribonucleoproteins.">Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.</a> Genome Biology. 18 (1), 92 (2017).</li><li>Park, K. E., 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+Gene+Knockin+in+Porcine+Somatic+Cells+Using+CRISPR/Cas+Ribonucleoproteins.">Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins.</a> International journal of molecular sciences. 217 (6), (2016).</li><li>Woo, J. 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=DNA-free+genome+editing+in+plants+with+preassembled+CRISPR-Cas9+ribonucleoproteins.">DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins.</a> Nature biotechnology. 33 (11), 1162-1164 (2015).</li><li>Harms, D. 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=Mouse+Genome+Editing+Using+the+CRISPR/Cas+System.">Mouse Genome Editing Using the CRISPR/Cas System.</a> Current protocols in human genetics. 83, 11-27 (2014).</li><li>Yang, H., Wang, H., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+genetically+modified+mice+using+CRISPR/Cas-mediated+genome+engineering.">Generating genetically modified mice using CRISPR/Cas-mediated genome engineering.</a> Nature protocols. 9 (8), 1956-1968 (2014).</li><li>Grompe, 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=Loss+of+Fumarylacetoacetate+Hydrolase+Is+Responsible+for+the+Neonatal+Hepatic-Dysfunction+Phenotype+of+Lethal+Albino+Mice.">Loss of Fumarylacetoacetate Hydrolase Is Responsible for the Neonatal Hepatic-Dysfunction Phenotype of Lethal Albino Mice.</a> Genes &amp; development. 7 (12), 2298-2307 (1993).</li><li>Paulk, N. K., 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=Adeno-associated+virus+gene+repair+corrects+a+mouse+model+of+hereditary+tyrosinemia+in+vivo.">Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo.</a> Hepatology. 51 (4), 1200-1208 (2010).</li></ol>

I passaggi più critici nella costruzione di plasmidi di donatore HMEJ sono: (1) selezione di sgRNA con alta efficienza di clivaggio del DNA e bassa distanza tra sito di taglio sgRNA e codone di arresto e costruzione (2) corretta del donatore HMEJ. CRISPR/Cas9-mediata cleavage su entrambi vettoriale di donatore del transgene (contenente siti di destinazione sgRNA e ~ 800 bp omologia braccia) e genoma mirato è necessario per l'integrazione mirata efficiente e precisa in vivo. I passaggi più critici della genera...

L'editing genomico precisa, mirata è spesso necessaria per la produzione di modelli animali geneticamente modificati e terapie cliniche. Molto sforzo è stato fatto per sviluppare varie strategie per efficiente genoma mirata modifiche, ad esempio l'apparato nucleasi a dita zinco (ZFN), nucleasi di trascrizione attivatore-come effettore (TALENs) e sistemi di CRISPR/Cas9. Queste strategie creare rotture a doppio filamento del DNA mirate (DSB) nel genoma e trarre vantaggio intrinseci sistemi di riparazione del DNA, come ricombinazione omologa (HR)1,2, basi-mediata fine unirsi (MMEJ)3 , 4 , 5e alla fine non-omologo unendo (NHEJ)6,7,8 per indurre l'integrazione mirata di transgeni1,9. La strategia basata su HR è attualmente il più comunemente usato genoma modificando l'approccio, che è molto efficiente in linee cellulari, ma non prontamente accessibile alle cellule di divisione a causa del relativo avvenimento limitata verso la fine della fase S/G2. Così, la strategia basata sulla non è applicabile per in vivo l'editing genomico. Recentemente, la strategia basata su NHEJ è stata sviluppata per efficiente gene knock-in mouse tessuti8. Tuttavia, il metodo basato su NHEJ introduce solitamente indels alle giunzioni, rendendo difficile generare l'editing genomico preciso, soprattutto quando si tenta di costruire in-frame fusione geni8. Integrazione mirata basata su MMEJ è capace di editing preciso genomico. Tuttavia, solo modestamente aumenta l'efficienza di integrazione mirata in precedenti relazioni5. Di conseguenza, migliorare l'efficienza della precisa integrazione mirata in vivo è urgente per applicazioni terapeutiche ampio3.
In un lavoro pubblicato recentemente, abbiamo dimostrato un fine omologia-mediata entrare (HMEJ)-base di strategia, che ha mostrato la massima efficienza di integrazione mirata a tutti segnalati strategie sia in vitro che in vivo10. Qui, descriviamo un protocollo per l'istituzione del sistema HMEJ, e anche la costruzione dei vettori di RNA (sgRNA) singolo-guida il gene di interesse e il donatore di targeting vettori harboring siti di destinazione sgRNA e ~ 800 bp di braccia di omologia (Figura 1) . In questo protocollo, descriviamo la procedura dettagliata per la generazione di topi knock-in DNA e brevi passi per l'integrazione mirata in tessuti in vivo. Inoltre, uno studio di proof-of-concept della strategia basata su HMEJ dimostrato la sua capacità di correggere Fah mutazione e salvare Fah- / - fegato guasto topi, che ulteriore ha rivelato il suo potenziale terapeutico.

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			<td height="40" style="height:40px;width:359px;">pX330</td>
			<td style="width:192px;">Addgene</td>
			<td style="width:131px;">42230</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:359px;">pAAV vector</td>
			<td style="width:192px;">Addgene</td>
			<td style="width:131px;">37083</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:359px;">pX260</td>
			<td style="width:192px;">Addgene</td>
			<td style="width:131px;">42229</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:359px;">AAV_Efs_hSpCas9_NLS_FLAG-SV40</td>
			<td style="width:192px;">Addgene</td>
			<td style="width:131px;">97307</td>
			<td style="width:524px;">AAV vector for encoding a human codon-optimized SpCas9 driven by EFs promoter</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:359px;">AAV_Actb HMEJ donor_U6_sgRNA_EF1a_GFP_polyA</td>
			<td style="width:192px;">Addgene</td>
			<td style="width:131px;">97308</td>
			<td style="width:524px;">HMEJ donor for fusing a p2A-mCherry reporter to mouse Actb. EGFP driven by EF1a promoter and U6-driven sgRNAs targeting Actb. AAV backbone.</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:359px;">AAV_Cdx2 HMEJ donor</td>
			<td style="width:192px;">Addgene</td>
			<td style="width:131px;">97319</td>
			<td style="width:524px;">HMEJ donor for fusing a p2A-mCherry reporter to mouse Cdx2.&#160;</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:359px;">Lipofectamine 3000 Transfection Reagent</td>
			<td style="width:192px;">Life Technology</td>
			<td style="width:131px;">L3000015</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Nuclease-Free Water</td>
			<td style="width:192px;">Life Technologies</td>
			<td style="width:131px;">AM9930</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Bbs I</td>
			<td style="width:192px;">New England Biolabs</td>
			<td style="width:131px;">R0539S</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">NEB Buffer 2</td>
			<td style="width:192px;">New England Biolabs</td>
			<td style="width:131px;">B7002S</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">T7 endonuclease I</td>
			<td style="width:192px;">New England Biolabs</td>
			<td style="width:131px;">M0302L</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">NEBuilder HiFi DNA Assembly Master Mix</td>
			<td style="width:192px;">New England Biolabs</td>
			<td style="width:131px;">E2621L</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Plasmid EndoFree-Midi Kit</td>
			<td style="width:192px;">Qiagen</td>
			<td style="width:131px;">12143</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">MMESSAGE MMACHINE T7 ULTRA</td>
			<td style="width:192px;">Life Technologies</td>
			<td style="width:131px;">AM1345</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">MEGACLEAR KIT 20 RXNS</td>
			<td style="width:192px;">Life Technologies</td>
			<td style="width:131px;">AM1908</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">MEGASHORTSCRIPT T7 KIT 25 RXNS</td>
			<td style="width:192px;">Life Technologies</td>
			<td style="width:131px;">AM1354</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Flaming/Brown Micropipette Puller</td>
			<td style="width:192px;">Sutter Instrument</td>
			<td style="width:131px;">P-97</td>
			<td style="width:524px;">&#160;Micropipette Puller (parameters: heat, 74; pull, 60; velocity, 80; time/delay, 200; pressure, 300)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Borosilicate glass</td>
			<td style="width:192px;">Sutter Instrument</td>
			<td style="width:131px;">B100-78-10</td>
			<td style="width:524px;">type of capillaries (outer diameter 1.0 mm, inner diameter 0.78 mm with filament)&#160;</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:359px;">FemtoJet microinjector</td>
			<td style="width:192px;">Eppendorf</td>
			<td style="width:131px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Freezing microtome</td>
			<td style="width:192px;">Leica</td>
			<td style="width:131px;">CM1950-Cryostat</td>
			<td style="width:524px;">thickness&#160;of&#160;40&#160;&#956;m&#160;for&#160;brain,&#160;10 &#956;m&#160;for&#160;liver</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Rabbit anti-mCherry</td>
			<td style="width:192px;">GeneTex</td>
			<td style="width:131px;"></td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Cy3-AffiniPure Goat Anti-Rabbit IgG</td>
			<td style="width:192px;">Jackson Immunoresearch</td>
			<td style="width:131px;"></td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">DMEM</td>
			<td style="width:192px;">Gibco</td>
			<td style="width:131px;">11965092</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:359px;">FBS</td>
			<td style="width:192px;">Gibco</td>
			<td style="width:131px;">10099141</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:359px;">NEAA</td>
			<td style="width:192px;">Gibco</td>
			<td style="width:131px;">11140050</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:359px;">Pen,Strep,Glutamine</td>
			<td style="width:192px;">Gibco</td>
			<td style="width:131px;">10378016</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:359px;">Gel Extraction Kit</td>
			<td style="width:192px;">Omega</td>
			<td style="width:131px;">D2500-02</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">FACS</td>
			<td>BD AriaII</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PMSG</td>
			<td>Ningbo Sansheng Medicine</td>
			<td>S141004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCG</td>
			<td>Ningbo Sansheng Medicine</td>
			<td>B141002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytochalasin B</td>
			<td>Sigma</td>
			<td>CAT#C6762</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KSOM+AA with D-Glucose and Phenol Red</td>
			<td>Millipore</td>
			<td>CAT#MR-106-D</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M2 Medium with Phenol Red</td>
			<td>Millipore</td>
			<td>CAT#MR-015-D</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mineral oil</td>
			<td>Sigma</td>
			<td style="width:131px;"></td>
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crispr/cas9-mediated targeted integration in vivo using a homology-mediated end joining-based strategy

Come una promettente piattaforma di editing del genoma, il sistema CRISPR/Cas9 ha un grande potenziale per la manipolazione genetica efficiente, soprattutto per l'integrazione mirata dei transgeni. Tuttavia, a causa della scarsa efficienza di ricombinazione omologa (HR) e le varie mutazioni indel di non-omologo fine unirsi (NHEJ)-basato su strategie in cellule di divisione, in vivo genome pubblica rimane una grande sfida. Qui, descriviamo una fine omologia-mediata entrare (HMEJ)-CRISPR/Cas9 sistema efficiente in vivo precisa mirata integrazione basato. In questo sistema, il genoma mirato e il donatore vector contenente armi di omologia (~ 800 bp) fiancheggiato da destinazione di RNA (sgRNA) guida singola sequenze sono spaccate da CRISPR/Cas9. Questa strategia basata su HMEJ realizza integrazione efficiente transgene in zigoti del mouse, così come in epatociti in vivo. Inoltre, una strategia basata su HMEJ offre un approccio efficace per la correzione di fumarylacetoacetate idrolasi (Fah) mutazione negli epatociti e Salva Fah-deficit indotto da topi di insufficienza epatica. Presi insieme, concentrandosi su mirata integrazione, questa strategia basata su HMEJ fornisce uno strumento promettente per una varietà di applicazioni, inclusa la generazione di modelli animali geneticamente modificati e terapie geniche mirate.

Editing basato su HMEJ genomico in embrioni di topo: Per definire l'efficienza knock-del metodo basato su HMEJ in zigoti mouse, abbiamo consegnato Cas9 mRNA, sgRNA targeting del gene Cdx2 e il donatore HMEJ in zigoti del mouse, che è stato progettato per fondere un gene reporter p2A-mCherry al codone ultimo di Cdx2 gene (Figura 2A). Gli zigoti iniettati sviluppato in blastocisti nella cultura. Per valutare ...

Watch this Scientific Journal Video about CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy at JoVE.com

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

Il cluster regolarmente intervallate proteine brevi ripetizioni/CRISPR palindromi associato 9 (CRISPR/Cas9) sistema fornisce uno strumento promettente per l'ingegneria genetica e apre la possibilità di integrazione mirata dei transgeni. Descriviamo un fine omologia-mediata entrare (HMEJ)-base di strategia per DNA efficiente mirata integrazione in vivo e mirate terapie geniche utilizzando CRISPR/Cas9.

Integrazione mirata CRISPR/Cas9-mediata In Vivo usando una strategia basata su unendo fine omologia-mediata

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

Research

JoVE Journal

Genetics

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

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic caso Mutagenesi Utilizzando istone-miniSOG in<em&gt; C. elegans</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Ricerca

Genetica

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Mirata<em&gt; In Situ</em&gt; mutagenesi dell&#39;istone geni in germogliamento lievito

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

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

Usando una tecnica fluorescente PCR-capillare elettroforesi su gel al genotipo CRISPR / Cas9 mediata Knockout Mutants in un formato high-throughput

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Selezione-dipendente e generazione indipendente di CRISPR / Cas9-mediata Gene Knockouts nelle cellule di mammiferi

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Un protocollo per la produzione di vettori lentivirali integrasi-carenti per CRISPR/Cas9-mediata Knockout del Gene nelle cellule in divisione

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Tempo di replicazione del DNA utilizzando Zebrafish come un sistema di modello In Vivo di profilatura

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Sequenziamento di nuova generazione e bioinformatica Pipeline per valutare fattori determinanti genetici della malattia costituzionale mirati

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

Determinare il tasso di fertilizzazione dell'uovo di Bemisia tabaci utilizzando una tecnica citogenetica

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

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

Applicazione in vivo del sistema di controllo remoto per la manipolazione dell'espressione genica endogena

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

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Studio funzionale in vivo sulle varianti umane rare associate alla malattia utilizzando la Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...