Name: selection-dependent and independent generation of crispr/cas9-mediated gene knockouts in mammalian cells
Uploaded: 2017-06-16T15:00:00
Description: Watch this Scientific Journal Video about Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells at JoVE.com

Forfatterne vil gerne anerkende Gissell Sanchez, Megan Lee og Jason Estep for eksperimentel bistand, og Weifeng Gu og Xuemei Chen til deling af reagenser.

1. Identifikation af homologiregioner omkring den ønskede sletning BEMÆRK: Kun nødvendigt, hvis du bruger valgbaseret redigering. <ol><li> Vælg to regioner, oprindeligt 1,5-2 kb, på hver side af det ønskede sletningslokal, som vil fungere som homologearmer i HDR-skabelonen ( Figur 1A ). Identificer regioner, der mangler BsaI anerkendelsessteder på begge strenger (GGTCTC) for at lette kloning. Hvis BsaI-steder er uundgåelige, skal du bruge et alternativ...

<ol>
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CRISPR / Cas9-systemet har muliggjort effektiv generering af stabile genomiske modifikationer, hvilket giver et mere konsistent alternativ til andre transient manipulationsmetoder. Her har vi præsenteret to metoder til hurtig identifikation af CRISPR / Cas9 gen-knockouts i pattedyrcellelinier. Begge metoder kræver lille cellulært materiale, så test kan ske i tidlige stadier af klonalkultur, hvilket sparer tid og reagenser. For at øge effektiviteten af ​​begge metoder anbefaler vi at teste flere sgRNA&#39;er, da...

<html> Stabile genetiske ændringer giver en fordel i forhold til transiente metoder til cellulær forstyrrelse, som kan variere i deres effektivitet og varighed. Genomisk redigering er blevet mere og mere almindelig de seneste år som følge af udviklingen af ​​målspecifikke nukleaser, såsom zinkfinger-nukleaser 1 , 2 , 3 , 4 , 5 , transkriptionsaktivator-lignende effektor-nucleaser (TALEN&#39;er) 6 , 7 , 8 , 9 Og RNA-styrede nucleaser afledt fra de klyngede, regelmæssigt interspaced korte palindromiske gentagelser (CRISPR) system 10 . CRISPR / Cas9-redigeringsmaskinen er tilpasset et immunsystem, som bakterier og archaea bruger til at forsvare mod virusinfektionerAss = &quot;xref&quot;&gt; 11 , 12 , 13 . I denne proces inkorporeres korte 20-30 nt-fragmenter af invaderende virussekvens i et genomisk locus som &quot;afstandsstykker&quot; flankeret af gentagende enheder 14 , 15 . Efterfølgende transkription og RNA-behandling genererer små CRISPR-associerede RNA&#39;er 16 (crRNA&#39;er), der sammen med et transaktiverende crRNA 17 (tracrRNA) samler med effektor Cas9 endonuclease. CrRNA&#39;erne tilvejebringer således specificitet til Cas9-målretning, styring af komplekset til spaltning af komplementære virale DNA-sekvenser og forebyggelse af yderligere infektioner 18 , 19 . Enhver &quot;protospacer&quot; -sekvens i det målrettede DNA kan tjene som kilden til crRNA, så længe den er direkte 5 &#39;til et kort protospacer tilstødende motiv (PAM), NGG i tilfælde af S. pyogenes Cas9 <sUp class = &quot;xref&quot;&gt; 20. Fraværet af PAM-sekvensen nær afstandsstykket i værtsens CRISPR-locus skelner mellem selv og ikke-selv, hvilket forhindrer målretning af værten. På grund af dets universalitet og fleksibilitet er dette biologiske system blevet kraftigt tilpasset til genomisk redigering, således at næsten ethvert PAM-tilstødende DNA-sted kan målrettes. I denne version smeltede en yderligere modifikation crRNA&#39;et og tracrRNA&#39;et i en enkelt guide RNA (sgRNA) komponent, der blev indlæst i Cas9 proteinet 21 . Ved ekspression af Cas9 og et sgRNA i eukaryote celler spalter Cas9-proteinet begge DNA-tråde på det målrettede sted. I fravær af en egnet region af homolog sekvens fikserer cellen denne pause via ikke-homolog end-sammenføjning (NHEJ) 22 , 23 , 24 , som typisk introducerer små deletioner eller sjældent insertioner. Når du målretter mod en åbenLæserammen fører reparationen sandsynligvis til en translationsramme, der frembringer et ikke-funktionelt proteinprodukt. I modsætning hertil kan cellen, når den er forsynet med en eksogen template med store områder af homologi, rette dobbeltstrengspausen ved hjælp af homologi-rettet reparation 25 , 26 . Denne rute tillader større præcise deletioner, udskiftninger eller insertioner i genomet, kombineret med indførelsen af ​​excisable markeringsmærker 27 . Her præsenterer vi protokoller til generering af knockout-cellelinjer ved hjælp af en af ​​disse to CRISPR / Cas9 metoder ( figur 1A ). NHEJ-tilgangen anvender et enkelt sgRNA-cut-site og selektionsafhængig screening, og kræver derfor lidt forudgående forberedelse. Ved brug af denne metode skal guide RNA&#39;er komplementære til exoner nær 5&#39;-enden af ​​transkriptet, som mest sandsynligt frembringer en knockout, skal udformes. Siden modificatIoner til genomet i dette tilfælde er små, screening for knockout-kloner er baseret på prikblots, hvor proteinproduktet vurderes på høj gennemstrømningsmåde. Vi bruger generationen af ​​ELAV-lignende 1 protein (ELAVL1) knockout linjer som et eksempel. Den anden tilgang er baseret på homologi-rettet reparation (HDR) og bruger to sgRNA-cut-sites, der spænder over genet eller regionen af ​​interesse, hvilket tillader sletninger af titusvis af kb. Et plasmid med to regioner af homologi, der flankerer spaltningsstederne, tilvejebringer en erstatningsskabelon ( figur 1B ), der indfører en selekterbar resistensmarkør, som øger effektiviteten af ​​knockout-generationen. Denne metode kan også tilpasses til at indføre genmodifikationer med ordentligt udformede homologi arme. I dette tilfælde muliggør integrationen af ​​et nyt DNA-fragment PCR-baseret screening ( figur 1C ). Her bruger vi generationen af ​​Pumilio RNA bindende familiemedlem 2 (PUM2) knockout linjer som et eksempel.

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">Competent E. coli cells</td>
			<td style="width:95px;"></td>
			<td style="width:120px;"></td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">plasmid prep kit</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pSpCas9(BB) plasmid</td>
			<td>Addgene</td>
			<td>42230</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BbsI enzyme</td>
			<td>ThermoFisher</td>
			<td>FD1014</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T7 DNA ligase</td>
			<td>NEB</td>
			<td>M0318L</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tango Buffer</td>
			<td>ThermoFisher</td>
			<td>BY5</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PlasmidSafe exonuclease</td>
			<td>Epicentre</td>
			<td>E3105K</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Q5 hot start high fidelity polymerase</td>
			<td>NEB</td>
			<td>M0494A</td>
			<td>HA amplification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC19-BsaI</td>
			<td></td>
			<td></td>
			<td>Modified pUC19 plasmid, mutated existing BsaI site and inserted two outward facing BsaI sites after BamHI/EcoRI digestion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pGolden-Neo</td>
			<td>Addgene</td>
			<td>51422</td>
			<td>Resistance cassette</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pGolden-Hygro</td>
			<td>Addgene</td>
			<td>51423</td>
			<td>Resistance cassette</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BsaI enzyme</td>
			<td>NEB</td>
			<td>R3535S</td>
			<td>Homology arm contruction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T7 DNA ligase</td>
			<td>NEB</td>
			<td>M0318L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PlasmidSafe exonuclease</td>
			<td>Epicentre</td>
			<td>E3105K</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Toothpicks</td>
			<td></td>
			<td></td>
			<td>bacterial PCR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taq polymerase</td>
			<td></td>
			<td></td>
			<td>bacterial colony PCR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEK293 human cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Corning</td>
			<td>MT35010CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Strep (opt.)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 well plates</td>
			<td>BioLite</td>
			<td>12556004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TransIT-LTI Transfection Reagent</td>
			<td>Mirus</td>
			<td>MIR2300</td>
			<td>for lipofection only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM</td>
			<td>ThermoFisher</td>
			<td>31985062</td>
			<td>for lipofection only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">G418 (Neomycin)</td>
			<td>Sigma Aldrich</td>
			<td>A1720-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hygromycin</td>
			<td>Sigma Aldrich</td>
			<td>H3274-250MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well plates</td>
			<td>ThermoFisher</td>
			<td>12556008</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Passive Lysis Buffer, 5x</td>
			<td>Promega</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">1xSDS loading buffer</td>
			<td></td>
			<td></td>
			<td>recipe decribed in protocol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nitrocellulose Membrane</td>
			<td>Bio-Rad</td>
			<td>162-0115</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TBST</td>
			<td></td>
			<td></td>
			<td>recipe decribed in protocol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dehydrated milk</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SuperSignal West Dura Extended Duration Substrate</td>
			<td>ThermoFisher</td>
			<td>34075</td>
			<td>for HRP-conjugated secondary antibodies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Extracta DNA prep for PCR</td>
			<td>Quantabio</td>
			<td>95091-025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KOD polymerase</td>
			<td>Novagen</td>
			<td>71316</td>
			<td></td>
		</tr>
	</tbody>
</table>

selection-dependent and independent generation of crispr/cas9-mediated gene knockouts in mammalian cells

CRISPR / Cas9 genomteknik systemet har revolutioneret biologi ved at tillade præcis genom redigering med lidt indsats. Guided af en enkelt guide RNA (sgRNA), der giver specificitet, spalter Cas9-proteinet begge DNA-tråde på det målrettede sted. DNA-pause kan udløse enten ikke-homolog slutningen (NHEJ) eller homologi-rettet reparation (HDR). NHEJ kan introducere små sletninger eller insertioner, der fører til rammeskift mutationer, mens HDR muliggør større og mere præcise forstyrrelser. Her præsenterer vi protokoller til generering af knockout-cellelinjer ved at kombinere etablerede CRISPR / Cas9 metoder med to muligheder for downstream-udvælgelse / screening. NHEJ-tilgangen anvender et enkelt sgRNA-cut-site og selektionsuafhængig screening, hvor proteinproduktion vurderes ved prik-immunoblot på en høj gennemstrømningsmåde. HDR-fremgangsmåden anvender to sgRNA-cut-sites, der spænder over genet af interesse. Sammen med en leveret HDR-skabelon kan denne metode opnå sletningAf titus kb, hjulpet af den indsatte valgbare resistansmarkør. De relevante applikationer og fordele ved hver metode diskuteres.

Til generering af ELAVL1 knockout linjer var et robust antistof tilgængeligt, så redigering ved anvendelse af enkelt sgRNA&#39;er ( Figur 1A , venstre) blev udført efterfulgt af prikimmunoblot. Tre sgRNA&#39;er blev transficeret uafhængigt for at sammenligne effektiviteter og at udelukke målvirkninger i de resulterende kloner. Efter opsamling og blotting af cellelysater fra klonale populationer på to nitrocellulosemembraner blev blottene probet for bå...

Watch this Scientific Journal Video about Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells at JoVE.com

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells (Video) | JoVE

Nylige fremskridt i evnen til at genetisk manipulere somatiske cellelinier har stort potentiale for grundlæggende og anvendt forskning. Her præsenterer vi to metoder til CRISPR / Cas9 genereret knockout produktion og screening i pattedyrcellelinjer, med og uden brug af selekterbare markører.

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 ...

Research

JoVE Journal

Genetics

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

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures (Video) | JoVE

Parallel Måling af døgnrytmen ur genekspression og hormon sekretion i Human primære cellekulturer

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus (Video) | JoVE

Opdræt og dobbelt-strenget RNA-medieret Gene Knockdown i Skjul Beetle,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

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

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

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 ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells (Video) | JoVE

En standardmetode til at undersøge ejendommen mutagenicitet som en funktion af punkt Mutation reparation katalyseret af CRISPR/Cas9 og SsODN i humane celler

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning (Video) | JoVE

Generation af en gen-forstyrret Streptococcus mutans stamme uden Gene kloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

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

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy (Video) | JoVE

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 ...

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

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

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 ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio (Video) | JoVE

Effektiv produktion og identifikation af CRISPR/Cas9-genereret gen Knockouts i Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells (Video) | JoVE

Formaldehyd-assisteret Isolation af regulerende elementer til foranstaltning kromatin tilgængelighed i pattedyrceller

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9 (Video) | JoVE

Højeffektive gen afbrydelse af Murine og menneskelige hæmatopoietisk stamceller ved CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...