Name: a rapid and facile pipeline for generating genomic point mutants in c. elegans using crispr/cas9 ribonucleoproteins
Uploaded: 2018-04-30T15:00:00
Description: Watch this Scientific Journal Video about A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins at JoVE.com

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Alle dyr omsorg og eksperimentelle prosedyrer fulgte retningslinjen fra National Institutes of Health og dyr institusjon og bruk Committee (IACUC) ved University of Michigan. Bruk RNase-free løsninger og Pipetter tips i protokollen. Rengjør arbeidsområde, Pipetter, rør, og sentrifuge med RNase dekontaminering løsning etter produsent retningslinjene (se Materialer tabell).
1. sgRNA Målvalg
<ol><li>Ved hjelp av en webleser, åpne websiden: http://crispor.tefor.net<sup cl...

<ol>
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CRISPR-Cas9 systemet er et kraftig og effektiv verktøy for å presist endre genomet av modellen organismer. Her viser vi at chimeric sgRNAs20 kombinert med ssODN HDR kan høyeffektive generering av genomisk punkt mutasjoner i C. elegans. Viktigere, viser vi at RNP levering produserer høy redigering effektivitet når fluorescens som en co markøren, opplyser det enkel og pålitelig teknikken.
Fleste av CRISPR-Cas9 C. elegans genomet engineering er avh...

Nyere teknologiske fremskritt har radikalt forvandlet og akselerert evnen til å nøyaktig ingeniør genomer. Spesielt har CRISPR-Cas9 systemet, som er avhenger av RNA-guidede endonuclease Cas9 å indusere en dobbel strand pause (DSB) nær målet sekvensen av interesse, vært mye brukt nøyaktig ingeniør genomet av flertallet av modell i biomedisinsk forskning1,2,3,4. Betydelig, bruk av CRISPR-Cas9 har ulåst genomet redigering selv i vanskelige arter som C. elegans5. Uansett art, generere punkt mutasjoner med CRISPR-Cas9 basert genomet redigering system avhengig tre kjerne-komponenter: 1) Cas9 endonuclease, 2) en enkelt hjelpelinje RNA (sgRNA) som leder Cas9 endonuclease en mål-sekvens, og 3) brukeren utformet homologi-rettet reparasjon (HDR) mal som inneholder ønsket edit(s) interesse2.
Det finnes flere metoder som kan brukes til å introdusere rettet mot sgRNA og Cas9 nuclease i cellene plasmider, RNA og viral basert levering metoder6. Nylig direkte levering av pre-kompleksbundet sgRNA-Cas9 Ribonucleoproteins (RNPs) har dukket opp som en kraftfull og effektiv verktøyet i CRISPR-Cas9-baserte genomet redigering7. Direkte levering av pre-kompleksbundet CRISPR-Cas9-RNPs har flere forskjellige fordeler, nemlig: 1) RNPs omgå behovet for mobilnettet transkripsjon og oversettelse, 2) RNPs fjernes raskt, som kan øke spesifisitet ved å redusere tilgjengelig tid for off-målet cleavage og 3) RNPs inneholder ingen fremmede DNA/RNA elementer som omgår innføring av ikke-innfødte sekvenser i vert genomet gjennom tilfeldige integrering. Disse attributtene sannsynlig utgjør sammen en kortvarig briste på målet CRISPR redigering samtidig minimere off-målet effekter.
Vi beskriver en enkel og effektiv protokoll for områdespesifikke genomisk endringer i C. elegans. Denne protokollen inkluderer målretting sgRNA og enkelt strandet oligonucleotide (ssODN) HDR mal tegning, sgRNA-Cas9 RNP complexing og levering, og en genotyperingteknologi strategi for utvetydig identifikasjon av riktig redigerte dyr. Bruker denne strategien, ikke bare kan de områdespesifikke endringene gjenopprettes, men andre ikke-spesifikke indel mutasjoner kan også gjenopprettes. Dermed vår strategi tillater generering av en allel serie med en enkelt strategi, der både mono-allel, bi-allel og indel mutanter kan genereres i F1 generasjon.

<table border="0" cellpadding="0" cellspacing="0" width="1144">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">CRISPRevolution sgRNA&#160; EZ Kit</td>
			<td style="width:216px;">Synthego Inc.</td>
			<td style="width:344px;"></td>
			<td style="width:227px;">chimeric sgRNA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nuclease-free TE</td>
			<td>Synthego Inc.</td>
			<td>provided with the sgRNA kit EZ kit</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nuclease-free water</td>
			<td>Synthego Inc.</td>
			<td>provided with the sgRNA kit EZ kit</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">4 nmole Ultramer DNA Oligo</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>ssODN HDR template</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alt-R S.p. HiFi Cas9 Nuclease 3NLS</td>
			<td>Integrated DNA Technologies</td>
			<td>1078728</td>
			<td>Cas9 protein</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">pCFJ90&#160;</td>
			<td>Addgene</td>
			<td>19327</td>
			<td>Pmyo-2::mCherry Marker Plasmid</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNA Clean &#38; Concentrator</td>
			<td>Zymo Research</td>
			<td>D4004</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Zymoclean Gel DNA Recovery Kit</td>
			<td>Zymo Research</td>
			<td>D4002</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Q5 Hot Start High-Fidelity 2X Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0494L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Proteinase K</td>
			<td>Sigma</td>
			<td>P2308</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass Borosilicate Glass Micropipettes</td>
			<td>Sutter Instruments</td>
			<td>BF100-78-10</td>
			<td>OD: 1.0mm. ID: 0.78mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trizma Hydrochloride</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MgCl2</td>
			<td>Sigma</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NP-40</td>
			<td>Sigma</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RNaseZap Decontamination Solution</td>
			<td>Fisher Scientific</td>
			<td>AM9780</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid and facile pipeline for generating genomic point mutants in c. elegans using crispr/cas9 ribonucleoproteins

Gruppert regelmessig avbrutt palindromic gjentakelser (CRISPR) - CRISPR - tilknyttede protein 9 (Cas9) prokaryote adaptive immunsystemet forsvar har vært medforfatter valgt som et kraftig verktøy for presis eukaryote genomet engineering. Her presenterer vi en rask og enkel metode bruke chimeric enkelt guide RNAs (sgRNA) og CRISPR-Cas9 Ribonucleoproteins (RNPs) for effektiv og presis generering av genomisk punkt mutasjoner i C. elegans. Vi beskriver en rørledning for sgRNA Målvalg, homologi-rettet reparasjon (HDR) mal tegning, CRISPR-Cas9-RNP complexing og levering og en genotyperingteknologi strategi som gir robust og rapid identifikasjon av riktig redigerte dyr. Vår tilnærming ikke bare tillater den lettvinte generasjon og identifikasjon av ønsket genomisk punkt mutant dyr, men også forenkler påvisning av andre komplekse indel alleler i ca 4-5 dager med høy effektivitet og en redusert screening arbeidsmengde.

Mutasjoner i menneskelig superoxide dismutase 1 (SOD1) utgjør ~ 10-20% av familiær amyotrofisk lateral sklerose, en ødeleggende nevrodegenerative sykdommer som alltid fører til lammelse og død17. Menneskelige TORV-1 er et evolusjonært bevarte protein deler 55% identitet og 70% likhet med C. elegans TORV-1 protein (figur 1B). For å demonstrere den enkelhet, gjennomførbarhet og effektivitet av CRISPR-Cas9 RNP-ba...

Watch this Scientific Journal Video about A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins at JoVE.com

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

En rask og lettvint rørledning for generering Genomic punkt mutanter i C. elegans bruker CRISPR/Cas9 Ribonucleoproteins

Her presenterer vi en metode for å ingeniør genomet C. elegans ved hjelp av CRISPR-Cas9 ribonucleoproteins og homologi avhengige reparasjon maler.

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

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A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

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