Name: adeno-associated virus-mediated delivery of crispr for cardiac gene editing in mice
Uploaded: 2018-08-02T13:00:00
Description: Watch this Scientific Journal Video about Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice at JoVE.com

U.R. è supportato dagli Stati Uniti istituti nazionali di sovvenzioni di salute (R01HL116546 e AR064241 R01).

Gli animali utilizzati in questo protocollo sono stati mantenuti a The Ohio State University laboratorio le risorse animali conformemente alle linee guida sull'uso degli animali. Tutti gli studi sugli animali sono stati autorizzati dal istituzionale Animal Care, uso e Comitato di revisione della Ohio State University.
1. progettazione e clonazione del gRNAs nel vettore CRISPR
<ol><li>Selezionare 2 gRNAs targeting distrofina introne 20 e introne 23 (i20 e i23) per SaCas9: i20: 5'-GGGCGTTGAAAT...

<ol>
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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annotation+and+Classification+of+CRISPR-Cas+Systems.">Annotation and Classification of CRISPR-Cas Systems.</a> Methods Mol Biol. 1311, 47-75 (2015).</li><li>Zetsche, 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=Cpf1+is+a+single+RNA-guided+endonuclease+of+a+class+2+CRISPR-Cas+system.">Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.</a> Cell. 163, 759-771 (2015).</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, 819-823 (2013).</li><li>Mali, P., 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=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339, 823-826 (2013).</li><li>Lieber, M. 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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=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nature protocols. 8, 2281-2308 (2013).</li><li>Zhang, X., Wang, J., Cheng, Q., Zheng, X., Zhao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+gene+regulation+by+CRISPR-ddCpf1.">Multiplex gene regulation by CRISPR-ddCpf1.</a> Cell discovery. 3, 17018 (2017).</li><li>Zhang, X. H., Tee, L. Y., Wang, X. G., Huang, Q. S., Yang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Off-target+Effects+in+CRISPR/Cas9-mediated+Genome+Engineering.">Off-target Effects in CRISPR/Cas9-mediated Genome Engineering.</a> Molecular therapy. Nucleic acids. 4, e264 (2015).</li><li>Xu, 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=CRISPR-mediated+genome+editing+restores+dystrophin+expression+and+function+in+mdx+mice.">CRISPR-mediated genome editing restores dystrophin expression and function in mdx mice.</a> Mol Ther. 24, 564-569 (2015).</li><li>Long, 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=Postnatal+genome+editing+partially+restores+dystrophin+expression+in+a+mouse+model+of+muscular+dystrophy.">Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy.</a> Science. 351, 400-403 (2016).</li><li>Long, C. 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=Prevention+of+muscular+dystrophy+in+mice+by+CRISPR/Cas9-mediated+editing+of+germline+DNA.">Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA.</a> Science. 345, 1184-1188 (2014).</li><li>Nelson, C. 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=In+vivo+genome+editing+improves+muscle+function+in+a+mouse+model+of+Duchenne+muscular+dystrophy.">In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy.</a> Science. 351, 403-407 (2016).</li><li>Ousterout, D. G. K., Thakore, A. M., Majoros, P. I., Reddy, T. E. W. H., Gersbach, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+CRISPR/Cas9-based+genome+editing+for+correction+of+dystrophin+mutations+that+cause+Duchenne+muscular+dystrophy.">Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy.</a> Nat Commun. 6, 6244 (2015).</li><li>Tabebordbar, 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=In+vivo+gene+editing+in+dystrophic+mouse+muscle+and+muscle+stem+cells.">In vivo gene editing in dystrophic mouse muscle and muscle stem cells.</a> Science. 351, 407-411 (2016).</li><li>Bengtsson, N. 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=Muscle-specific+CRISPR/Cas9+dystrophin+gene+editing+ameliorates+pathophysiology+in+a+mouse+model+for+Duchenne+muscular+dystrophy.">Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy.</a> Nat Commun. 8, 14454 (2017).</li><li>Hoffman, E. P., Brown, R. H., Kunkel, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin:+the+protein+product+of+the+Duchenne+muscular+dystrophy+locus.">Dystrophin: the protein product of the Duchenne muscular dystrophy locus.</a> Cell. 51, 919-928 (1987).</li><li>Nowak, K. J., Davies, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duchenne+muscular+dystrophy+and+dystrophin:+pathogenesis+and+opportunities+for+treatment.">Duchenne muscular dystrophy and dystrophin: pathogenesis and opportunities for treatment.</a> EMBO reports. 5, 872-876 (2004).</li><li>Mendell, J. 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=Evidence-based+path+to+newborn+screening+for+Duchenne+muscular+dystrophy.">Evidence-based path to newborn screening for Duchenne muscular dystrophy.</a> Annals of neurology. 71, 304-313 (2012).</li><li>Mendell, J. R., Lloyd-Puryear, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Report+of+MDA+muscle+disease+symposium+on+newborn+screening+for+Duchenne+muscular+dystrophy.">Report of MDA muscle disease symposium on newborn screening for Duchenne muscular dystrophy.</a> Muscle Nerve. 48, 21-26 (2013).</li><li>Spurney, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyopathy+of+Duchenne+muscular+dystrophy:+current+understanding+and+future+directions.">Cardiomyopathy of Duchenne muscular dystrophy: current understanding and future directions.</a> Muscle Nerve. 44, 8-19 (2011).</li><li>Moriuchi, T., Kagawa, N., Mukoyama, M., Hizawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autopsy+analyses+of+the+muscular+dystrophies.">Autopsy analyses of the muscular dystrophies.</a> Tokushima J Exp Med. 40, 83-93 (1993).</li><li>McNally, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+in+the+therapy+of+cardiomyopathy+in+muscular+dystrophy.">New approaches in the therapy of cardiomyopathy in muscular dystrophy.</a> Annual review of medicine. 58, 75-88 (2007).</li><li>Baker, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approvals,+Submission,+and+Important+Labeling+Changes+for+US+Marketed+Pharmaceuticals.">Approvals, Submission, and Important Labeling Changes for US Marketed Pharmaceuticals.</a> Hosp Pharm. 52, 306-307 (2013).</li><li>Baker, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eteplirsen.">Eteplirsen.</a> Hosp Pharm. 52, 302-305 (2017).</li><li>Sicinski, P., 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+molecular+basis+of+muscular+dystrophy+in+the+mdx+mouse:+a+point+mutation.">The molecular basis of muscular dystrophy in the mdx mouse: a point mutation.</a> Science. 244, 1578-1580 (1989).</li><li>Xu, 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=CRISPR-mediated+Genome+Editing+Restores+Dystrophin+Expression+and+Function+in+mdx+Mice.">CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice.</a> Molecular therapy : the journal of the American Society of Gene Therapy. 24, 564-569 (2016).</li><li>El Refaey, 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=In+Vivo+Genome+Editing+Restores+Dystrophin+Expression+and+Cardiac+Function+in+Dystrophic+Mice.">In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice.</a> Circ Res. 121, 923-929 (2017).</li><li>Tabebordbar, 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=In+vivo+gene+editing+in+dystrophic+mouse+muscle+and+muscle+stem+cells.">In vivo gene editing in dystrophic mouse muscle and muscle stem cells.</a> Science. 351, 407-411 (2016).</li><li>Pozsgai, E. R., Griffin, D. A., Heller, K. N., Mendell, J. R., Rodino-Klapac, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Sarcoglycan+gene+transfer+decreases+fibrosis+and+restores+force+in+LGMD2E+mice.">beta-Sarcoglycan gene transfer decreases fibrosis and restores force in LGMD2E mice.</a> Gene therapy. 23, 57-66 (2016).</li><li>Chicoine, L. 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=Plasmapheresis+eliminates+the+negative+impact+of+AAV+antibodies+on+microdystrophin+gene+expression+following+vascular+delivery.">Plasmapheresis eliminates the negative impact of AAV antibodies on microdystrophin gene expression following vascular delivery.</a> Mol Ther. 22, 338-347 (2014).</li><li>Chicoine, L. 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=Vascular+delivery+of+rAAVrh74.MCK.GALGT2+to+the+gastrocnemius+muscle+of+the+rhesus+macaque+stimulates+the+expression+of+dystrophin+and+laminin+alpha2+surrogates.">Vascular delivery of rAAVrh74.MCK.GALGT2 to the gastrocnemius muscle of the rhesus macaque stimulates the expression of dystrophin and laminin alpha2 surrogates.</a> Mol Ther. 22, 713-724 (2014).</li><li>Vouillot, L., Thelie, A., Pollet, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+T7E1+and+surveyor+mismatch+cleavage+assays+to+detect+mutations+triggered+by+engineered+nucleases.">Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases.</a> G3. 5, 407-415 (2015).</li><li>Tsai, S. Q., 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=GUIDE-seq+enables+genome-wide+profiling+of+off-target+cleavage+by+CRISPR-Cas+nucleases.">GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.</a> Nat Biotechnol. 33, 187-197 (2015).</li></ol>

In questo protocollo, abbiamo dettaglio tutti i passi necessari per raggiungere in vivo gene cardiaco modifica in topi postnatali utilizza il recapito rAAVrh.74-mediata di SaCas9 e due gRNAs. Come precedentemente osservato, questo approccio può essere utilizzato per ripristinare l'espressione di distrofina in topi distrofici come descritto nel nostro lavoro27, ma potrebbero anche permettere gli studi di genetica inversa rapida dei geni legati al cardiaco in topi senza il lungo processo d...

Il cluster, regolarmente intercapedine, breve palindromi ripetere (CRISPR) tecnologia rappresenta il più recente avanzamento in ingegneria del genoma e ha rivoluzionato la pratica corrente della genetica in cellule e degli organismi. Editing basato su CRISPR genomico utilizza una sola guida RNA (gRNA) per dirigere un'endonucleasi associata CRISPR come CRISPR-associated protein 9 (Cas9) e Cpf1 ad un target di DNA genomico che è complementare in sequenza la protospacer codificati da gRNA con un protospacer specifici motivi adiacenti (PAM)1,2. Il PAM varia a seconda della specie batterica del gene Cas9 o Cpf1. Il più comunemente usata nucleasi Cas9 derivata da Streptococcus pyogenes (SpCas9) riconosce una sequenza di PAM di NGG che si trova direttamente a valle della sequenza dell'obiettivo nel DNA genomico, sul filo del non-bersaglio. Presso il sito di destinazione, le proteine Cas9 e Cpf1 generano una rotture a doppio filamento (DSB), che sono poi ripristinata via intrinseci meccanismi di riparazione del DNA cellulari tra cui unirsi non-omologo fine (NHEJ) e riparazione omologia-diretto (HDR) vie3, 4. In assenza di un modello di DNA per ricombinazione omologa, il DSB è principalmente riparato da pathway NHEJ errori, che possono provocare gli inserimenti o eliminazioni (indels) di nucleotidi causando mutazioni frameshift, se l'organo di conciliazione sono stato creato nella codificazione regione di un gene5,6. Così, il sistema CRISPR è stato ampiamente utilizzato per progettare le mutazioni di perdita-de-funzione in vari modelli cellulari e interi organismi, al fine di studiare la funzione di un gene. Inoltre, il cataliticamente inattivo Cas9 e Cpf1 sono stati sviluppati per trascrizionalmente ed epigeneticamente modulare l'espressione di geni bersaglio7,8.
Più recentemente, sia SpCas9 e SaCas9 (derivato da Staphylococcus aureus) sono stati imballati in vettori ricombinanti virus adeno-associato (rAAV) per la correzione genica postnatale nel modello animale di malattie umane, in particolare, Duchenne distrofia muscolare (DMD)9,10,11,12,13,14,15. DMD è una malattia recessiva legata al X fatale causata da mutazioni nel gene DMD , che codifica per la proteina distrofina16,17, che interessa circa uno in 3500 nati maschi secondo screening neonatale18 , 19. è caratterizzata da debolezza muscolare progressiva e lo spreco. I pazienti DMD di solito perdono la capacità di camminare tra i 10 e i 12 anni di età e morire di insufficienza respiratoria e/o cardiaco all'età di 20-30 anni20. In particolare, la cardiomiopatia si sviluppa in > 90% dei pazienti DMD e rappresenta la principale causa di morte in pazienti DMD21,22. Anche se Deflazacort (un farmaco anti-infiammatorio) ed Eteplirsen (un esone che salta medicina per saltare essone 51) recentemente sono stati approvati per la DMD da Food and Drug Administration (FDA)23,24, nessuno di questi trattamenti correggere il difetto genetico a livello di DNA genomico. Il topo mdx, che trasporta una mutazione di punto all'esone 23 del gene della distrofina, è stato ampiamente usato come un DMD modello25. Inoltre, abbiamo in precedenza dimostrato che l'espressione di distrofina funzionale è stato restaurato da omissione in-struttura del DNA genomic che coprono gli essoni 21, 22 e 23 nella muscolatura scheletrica di topi mdx utilizzando intramuscolare annuncio-SpCas9/gRNA consegna9 e nei muscoli del cuore di topi mdx/utrofina+ /- mediante consegna di rAAVrh.74-SaCas9/gRNA endovenosa ed intraperitoneale26.
In questo protocollo, descriviamo in dettaglio ogni passo nel gene cardiaco postnatale di editing per ripristinare la distrofina in topi mdx/utrofina+ /- utilizzando rAAVrh.74-SaCas9/gRNA vettori, dalla progettazione di gRNA alla distrofina analisi nelle sezioni del muscolo di cuore.

<table>
	<tbody>
		<tr>
			<td>Alexa Fluor 555 goat anti-rabbit IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21428</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Sigma-Aldrich</td>
			<td>E1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio Safety Cabinets</td>
			<td>Thermo Fisher Scientific</td>
			<td>1347</td>
			<td>1300 Series A2 Class II, Type A2</td>
		</tr>
		<tr>
			<td>BsaI</td>
			<td>New England BioLabs Inc</td>
			<td>RO535S</td>
			<td></td>
		</tr>
		<tr>
			<td>Competent cells</td>
			<td>Agilent Technologies</td>
			<td>200314</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dishes, 150mm</td>
			<td>Nest Scientific USA</td>
			<td>715001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>Leica CM3050S</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>MT10017CV</td>
			<td>Corning cellgro</td>
		</tr>
		<tr>
			<td>Dystrophin antibody</td>
			<td>Spring Bioscience</td>
			<td>E2660</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast-Ion Midi Plasmdi Kits</td>
			<td>IBI Scientific</td>
			<td>IB47111</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>26-140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Unit, 0.45&#956;m</td>
			<td>Thermo Fisher Scientific</td>
			<td>166-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>GoTaq Master Mixes</td>
			<td>Promega</td>
			<td>M7122</td>
			<td></td>
		</tr>
		<tr>
			<td>High-speed plasmid mini kit</td>
			<td>IBI Scientific</td>
			<td>IB47102</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Abcam</td>
			<td>ab139501</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp 2239 Water Bath</td>
			<td>Thermo Fisher Scientific</td>
			<td>15-460-11Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp Heat Block</td>
			<td>Thermo Fisher Scientific</td>
			<td>11-718</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>Carl Zeiss Microscopy, LLC</td>
			<td></td>
			<td>LSM 780</td>
		</tr>
		<tr>
			<td>LightCycler 480 instrument</td>
			<td>Roche</td>
			<td>5015243001</td>
			<td>LightCycler 480 Instrument II</td>
		</tr>
		<tr>
			<td>Large Capacity Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>46-910</td>
			<td>Thermo Scientific Sorvall RC 6 Plus</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002445</td>
			<td>Sorvall Legend Micro 21R</td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotometers</td>
			<td>Thermo Scientific</td>
			<td>ND2000CLAPTOP</td>
			<td>NanoDrop&#8482; 2000/2000c</td>
		</tr>
		<tr>
			<td>Oligos for i20-F</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>CACCgGGCGTTGAAATTCTCATTAC</td>
		</tr>
		<tr>
			<td>Oligos for i20-R</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>AAAC GTAATGAGAATTTCAACGCCc;</td>
		</tr>
		<tr>
			<td>Oligos for i23-F</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>CACCgCACCGATGAGAGGGAAAGGTC</td>
		</tr>
		<tr>
			<td>Oligos for i23-R</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>GACCTTTCCCTCTCATCGGTGc</td>
		</tr>
		<tr>
			<td>Oligos</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep Density Gradient Medium</td>
			<td>Sigma-Aldrich</td>
			<td>D1556-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG8000</td>
			<td>Sigma-Aldrich</td>
			<td>89510-1kg-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Polysciences</td>
			<td>23966</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>P2308</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick-Seal centrifuge tube</td>
			<td>Beckman Coulter Inc</td>
			<td>342414</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Radiant SYBR Green Hi-ROX qPCR Kits</td>
			<td>Alkali Scientific</td>
			<td>QS2005</td>
			<td></td>
		</tr>
		<tr>
			<td>RevertAid RT Reverse Transcription Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K1691</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotor</td>
			<td>Beckman Coulter Inc</td>
			<td>337922</td>
			<td>Type 70Ti</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England BioLabs Inc</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Bio-rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter Inc</td>
			<td>8043-30-1192</td>
			<td>Optima le-80k</td>
		</tr>
		<tr>
			<td>Ultra centrifugal filter unit</td>
			<td>MilliporeSigma</td>
			<td>UFC510096</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Mounting Medium with DAPI</td>
			<td>Vector Laboratories, Inc</td>
			<td>H-1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

adeno-associated virus-mediated delivery of crispr for cardiac gene editing in mice

Il sistema cluster, regolarmente intercapedine, breve, palindromico di ripetere (CRISPR) ha notevolmente facilitato ingegneria genoma in cellule coltivate e gli organismi viventi da una grande varietà di specie. La tecnologia CRISPR è stata esplorata anche come approcci terapeutici per un certo numero di malattie umane. Dati di prova sono molto incoraggianti, come esemplificato da studi recenti che dimostrano la fattibilità e l'efficacia di gene editing basato su approccio terapeutico per la distrofia muscolare di Duchenne (DMD) utilizzando un modello murino. In particolare, iniezione endovenosa ed intraperitoneale del rh.74 di sierotipo del virus adeno-associato ricombinante (rAAV) (rAAVrh.74) ha permesso la consegna efficiente cardiaca di Staphylococcus aureus CRISPR-collegata della proteina 9 (SaCas9) e due Guida RNAs (gRNA) per eliminare una regione genomica con un codone mutante nell'esone 23 del gene Dmd del mouse. Questo stesso approccio è utilizzabile anche per battere il gene di interesse e studiare la loro funzione cardiaca nei topi postnatali quando il gRNA è progettato per la regione di codificazione del gene. In questo protocollo, vi mostriamo nel dettaglio come ingegnere vector rAAVrh.74-CRISPR e come raggiungere altamente efficiente consegna cardiaca in topi neonatali.

L'efficacia di gRNAs per indurre l'eliminazione della regione di DNA genomica di destinazione dovrebbe essere valutata in colture di cellule prima del confezionamento in rAAV per studi in vivo. A tal fine, il gRNAs e il SaCas9 esprimenti costrutti erano individulamente in cellule C2C12 e il DNA genomico è stato analizzato mediante PCR con primers fiancheggianti i siti di destinazione (Figura 1). Un prodotto PCR di ~ 500 bp indica l'eliminazione di successo d...

Watch this Scientific Journal Video about Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice at JoVE.com

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

Qui forniamo un protocollo dettagliato per effettuare in vivo gene cardiaco editing nei topi utilizzando Virus ricombinante Adeno-Associated (rAAV)-mediate consegna di CRISPR. Questo protocollo offre una strategia terapeutica di promessa per il trattamento di cardiomiopatia distrofica nella distrofia muscolare di Duchenne e può essere utilizzato per generare specifico knockout in topi postnatali.

Adeno-associato Virus-mediate consegna di CRISPR per Gene cardiaco nei topi di Editing

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Research

JoVE Journal

Medicine

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Ad alta efficienza di trasduzione delle cellule tumorali del fegato da adeno-associato ricombinante sierotipo Virus 3 Vettori

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

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Medicina

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Istituzione e Propagazione dei Tumori retinoblastoma umani in topi privi di sistema immunitario

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

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Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

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Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

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Bile Duct Ligation in topi: Induzione infiammatoria epatica Lesioni e fibrosi da colestasi ostruttiva

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

Utilizzando virus adeno-associati come strumento per studiare le barriere della retina nella malattia

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

Sviluppo di un Rat modello Alfa-sinucleina per la malattia di Parkinson mediante iniezione stereotassica di un ricombinante vettore virale adeno-associato

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Infarto del miocardio in topi neonati, un modello di rigenerazione cardiaca

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

Approccio del canale semicircolare posteriore per consegna del Gene dell'orecchio interno in Mouse neonatale

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of ...