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

Dette arbejde blev støttet af CAS strategiske prioritet Research Program (XDB02050007, XDA01010409), den nationale Hightech R & D Program (863 Program; 2015AA020307), National Natural Science Foundation of China (NSFC tilskud 31522037, 31500825, 31571509, 31522038), Kina ungdom tusinde talenter Program (til HY), bryde gennem projektet af kinesiske Academy of Sciences, projekt Shanghai City Udvalget for videnskab og teknologi (16JC1420202 til HY), Ministeriet for videnskab og teknologi i Kina (de fleste; 2016YFA0100500).

Alle procedurer, herunder animalske emner er blevet godkendt af den biomedicinske forskning etiske komité på Shanghai institutter for biologisk videnskab (CAS).
1. design af Donor plasmider
<ol><li>
Valg af sgRNA
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<li>Brug online CRISPR design værktøj til at forudsige sgRNAs på den indskyde region11,12,13,14,1...

<ol>
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De mest afgørende skridt i opbygningen af HMEJ donor plasmider er: (1) valg af sgRNA med høj DNA kavalergang effektivitet og lave afstand mellem sgRNA skæring websted og stop codon, og (2) en korrekt opførelse af HMEJ donor. CRISPR/Cas9-medieret kavalergang på begge transgen donor vektor (indeholdende sgRNA målwebsteder og ~ 800 bp homologi våben) og målrettede genom er nødvendige for effektivt og præcist målrettede integration in vivo. De mest kritiske trin i generation af knock-i mus ved hjælp af H...

Præcise, målrettede genom-redigering er ofte påkrævet for at producere genetisk modificerede dyremodeller og kliniske behandlinger. Stor indsats er gjort at udvikle forskellige strategier for effektiv målrettede genom-redigering, såsom zink nukleasen (ZFN), transkription aktivere-lignende effektor nukleaser (TALENs), og CRISPR/Cas9 systemer. Disse strategier skabe målrettede DNA dobbelt-strenget pauser (DSB) i genomet, og drage fordel af iboende DNA reparation systemer, såsom homologe rekombination (HR)1,2, microhomology-medieret ende sammenføjning (MMEJ)3 , 4 , 5, og ikke-homologe ende sammenføjning (NHEJ)6,7,8 for at fremkalde målrettede integration af transgener1,9. Den HR-baseret strategi er i øjeblikket mest almindeligt anvendte genom-redigering tilgang, som er meget effektiv i cellelinjer, men ikke let tilgængelige for ikke-dividere celler på grund af dens begrænsede forekomst i den sene S/G2 fase. Således, den HR-baseret strategi er ikke gældende for i vivo genom-redigering. For nylig, den NHEJ-baseret strategi er udviklet til effektiv gen banke i i mus væv8. Ikke desto mindre, den NHEJ-baseret metode normalt introducerer indels på vejkryds, hvilket gør det vanskeligt at generere præcis genom-redigering, især når de forsøger at konstruere i-frame fusion gener8. MMEJ-baserede målrettede integration er i stand til præcis genom-redigering. Det øger dog kun beskedent målrettede integration effektiviteten i tidligere rapporter5. Derfor, effektivisering af præcist målrettede integration i vivo er bydende nødvendigt for bred terapeutisk anvendelse3.
I en nyligt offentliggjort arbejde, vi viste en homologi-medieret ende sammenføjning (HMEJ)-baseret strategi, som viste den højeste målrettede integration effektivitet i alle rapporterede strategier både in vitro- og i vivo10. Her, vi beskriver en protokol om oprettelse af HMEJ systemet, og også opførelsen af single-guide RNA (sgRNA) vektorer målretning gen af interesse og donor vektorer husly sgRNA målwebsteder og ~ 800 bp homologi våben (figur 1) . I denne protokol beskriver vi også de detaljerede trin til generering af DNA knock-i mus og korte trin for målrettede integration i væv i vivo. Desuden viste en proof of concept undersøgelse af HMEJ-baseret strategien sin evne til at korrigere Fah mutation og redde Fah- / - leveren manglende mus, som yderligere afslørede dets terapeutiske potentiale.

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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>
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			<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>
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			<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>
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			<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>
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		<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>
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		<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>
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		<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>
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			<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>
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			<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>
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			<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>
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		<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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			<td height="40" style="height:40px;width:359px;">FemtoJet microinjector</td>
			<td style="width:192px;">Eppendorf</td>
			<td style="width:131px;"></td>
			<td></td>
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		<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>
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			<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>
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		<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>
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			<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>
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			<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>
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		<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>
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			<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>
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			<td height="26" style="height:26px;">FACS</td>
			<td>BD AriaII</td>
			<td></td>
			<td></td>
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		<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>
			<td style="width:524px;"></td>
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crispr/cas9-mediated targeted integration in vivo using a homology-mediated end joining-based strategy

Som en lovende genom redigering platform har CRISPR/Cas9-systemet et stort potentiale for effektiv genetisk manipulation, især for målrettede integration af transgener. Men på grund af den lave effektivitet af homologe rekombination (HR) og forskellige indel mutationer af ikke-homologe ende sammenføjning (NHEJ)-baserede strategier i ikke-dividere celler, i vivo genom-redigering er fortsat en stor udfordring. Her, vi beskriver en homologi-medieret ende sammenføjning (HMEJ)-baseret CRISPR/Cas9 system til effektiv i vivo præcist målrettede integration. I dette system, de målrettede genom og donor vektor indeholdende homologi arme (~ 800 bp) flankeret af enkelt guide RNA (sgRNA) mål sekvenser er kløvet af CRISPR/Cas9. Denne HMEJ-baseret strategi opnår effektiv transgen integration i mus zygotes samt i hepatocytter i vivo. Desuden en HMEJ-baseret strategi tilbyder en effektiv tilgang til korrektion af fumarylacetoacetate hydrolase (Fah) mutation i hepatocytter og redder Fah-vitaminmangel induceret leversvigt mus. Taget sammen, med fokus på målrettet integration, denne HMEJ-baseret strategi giver et lovende redskab for en bred vifte af applikationer, herunder generation af genetisk modificerede dyremodeller og målrettede genterapier.

HMEJ-baseret genom-redigering i mus embryoner: Hvis du vil definere knock-i effektiviteten af HMEJ-baseret metode i mus zygotes, leverede vi Cas9 mRNA, sgRNA rettet mod Cdx2 -gen og HMEJ donor til musen zygotes, som var designet til at lunte en p2A-mCherry reporter gen til den sidste codon af Cdx2 gen (figur 2A). De injicerede zygotes udviklet sig til blastocyster i kulturen. For at evaluere overvågningsakt...

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

De grupperede regelmæssigt interspaced korte palindromiske gentager/CRISPR forbundet protein 9 (CRISPR/Cas9) system giver et lovende redskab for genteknologi, og åbner op for muligheden for målrettede integration af transgener. Vi beskriver en homologi-medieret ende sammenføjning (HMEJ)-baseret strategi for effektiv DNA målrettet integration i vivo og målrettet genterapier bruger CRISPR/Cas9.

CRISPR/Cas9-medieret målrettede Integration In Vivo ved hjælp af en homologi-medieret End at deltage-baseret strategi

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 Tilfældig mutagenese ved anvendelse histon-miniSOG i<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 ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Målrettet<em&gt; I Situ</em&gt; Mutagenese af histon-gener i knopskydende gær

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

Under anvendelse af et fluorescerende PCR-kapillær gelelektroforese Teknik til genotype CRISPR / Cas9-medieret Knockout Mutanter i et format med høj kapacitet

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

Udvælgelsesafhængig og uafhængig generering af CRISPR / Cas9-medierede Gene Knockouts i Pattedyrceller

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

En protokol til produktion af Integrase-mangelfuld Lentiviral vektorer for CRISPR/Cas9-medieret gen Knockout i dividere celler

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

Profilering DNA replikation Timing ved hjælp af zebrafisk som en In Vivo modelsystem.

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

Målrettet næste generation Sequencing og bioinformatik rørledningen for at evaluere genetiske determinanter for forfatningsmæssige sygdom

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

Fastsættelsen af den æg befrugtning Bemisia tabaci ved hjælp af en cytogenetisk teknik

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

In vivo anvendelse af sen-kontrol-System for Manipulation af endogene genekspression

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

In vivo funktionel undersøgelse af sygdoms associerede sjældne humane varianter ved hjælp af Drosophila

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