Name: genome editing in mammalian cell lines using crispr-cas
Uploaded: 2019-04-11T12:30:00
Description: Watch this Scientific Journal Video about Genome Editing in Mammalian Cell Lines using CRISPR-Cas at JoVE.com

M.H.T. støttes av et byrå for vitenskap teknologi og Research felles rådet Office grant (1431AFG103), gir en National Medical Research Council (OFIRG/0017/2016), National Research Foundation gir (NRF2013-THE001-046 og NRF2013-THE001-093), en Departementet for utdanning Tier 1 grant (RG50-17 (S)), en oppstart gi fra Nanyang Technological University, og midler til internasjonale genetisk Engineering maskin (iGEM) konkurransen fra Nanyang Technological University.

1. design av sgRNAs
<ol><li>Velg en passende CRISPR-Cas-system.<ol>
<li>Først inspisere målregion for PAM sekvensene av alle Cas9 og Cas12a nucleases som har vist seg å fungere i pattedyrceller16,21-32. Fem brukte enzymer er gitt i tabell 1 sammen med deres respektive PAMs. Merk: i tillegg til endonucleases oppført i tabell 1, det finnes andre mindre brukte Cas enzymer som har blitt distribuert i pattedyrceller også, som en Cas9 nuclease fra Streptococcus thermop...

CRISPR-Cas systemet er en kraftig og revolusjonerende teknologi til ingeniør genomes og transcriptomes av planter og dyr. Mange bakteriell arter er funnet for å inneholde CRISPR-Cas-systemer, som kan være tilpasset genom og transcriptome engineering formål44. Selv om Cas9 endonuclease fra Streptococcus pyogenes (SpCas9) var den første enzymet distribueres vellykket i menneskeceller21,22,23<s...

Muligheten til å ingeniør genomet av enhver levende organisme har mange biomedisinsk og bioteknologisk programmer, for eksempel korrigering av sykdomsfremkallende mutasjoner, bygging av nøyaktige mobilnettet modeller for sykdom studier eller generering av landbruket vekster med ønskelig egenskaper. Siden begynnelsen av århundret, ulike teknologier er utviklet for genomet engineering i pattedyrceller, inkludert meganucleases1,2,3, sink finger nucleases4,5, eller transkripsjon aktivator som effektor nucleases (TALENs)6,7,8,9. Men er disse tidligere teknologiene vanskelig å programmet eller kjedelig å montere, og dermed hindrer deres utbredt adopsjon i forskning og industri.
De siste årene, de klynget interspaced regelmessig kort palindromic gjentar (CRISPR) - CRISPR-assosiert (Cas) systemet har dukket opp som en kraftig ny genomet engineering teknologi10,11. Opprinnelig en adaptive immunsystemet i bakterier, det har blitt deployert for genomet endring i planter og dyr, inkludert mennesker. Hovedårsak hvorfor CRISPR-Cas har fått så mye popularitet i så kort tid er at elementet som bringer den viktigste Cas-endonuclease, som Cas9 eller Cas12a (også kjent som Cpf1), til riktig sted i genomet er bare et kort stykke chimeric enkelt støttelinje RN En (sgRNA), som er enkelt å utforme og billig å syntetisere. Etter blir rekruttert til målområdet, Cas enzymet fungerer som et par molekylær saks og innstiftet bundet DNA med sin RuvC, HNH eller Nuc domener12,13,14. Resulterende dobbel strandet pause (DSB) repareres senere av cellene via enten ikke-homologe slutten med (NHEJ) eller homologi-rettet reparasjon (HDR) veien. I fravær av en reparasjon mal repareres DSB av feilutsatte NHEJ veien, som kan gi opphav til pseudo-tilfeldige innsetting eller sletting av nukleotider (indeler) på webområdet kutt, potensielt forårsake frameshift mutasjoner i protein-koding gener. Men i nærvær av en donor mal som inneholder endringene i DNA, er DSB reparert av Hi-Fi HDR veien. Vanlige typer donor maler inkluderer enkelt-strandet oligonucleotides (ssODNs) og plasmider. Tidligere brukes vanligvis hvis tiltenkte DNA endringene er små (for eksempel endring av en enkelt base-par), mens sistnevnte brukes vanligvis hvis man ønsker å sette inn en relativt lang sekvens (for eksempel koding sekvensen av en grønn fluorescerende protein eller GFP) i målet locus.
Endonuclease aktiviteten til Cas protein krever tilstedeværelse av en protospacer tilstøtende motiv (PAM) mål område15. PAM Cas9 er på 3 slutten av protospacer, PAM Cas12a (også kalt Cpf1) er på 5' slutten i stedet16. Cas-guiden RNA komplekse er ikke innføre en DSB hvis PAM er fraværende17. Derfor plasserer PAM en begrensning på genomisk stedene der en bestemt Cas-nuclease er i stand til å holde seg. Heldigvis viser Cas nucleases fra ulike bakterie-art vanligvis ulike PAM krav. Ved å integrere ulike CRISPR-Cas systemer i våre tekniske verktøykassen, kan vi derfor utvide omfanget av nettsteder som kan målrettes i et genom. Videre kan et naturlig Cas enzym være konstruert eller utviklet for å gjenkjenne alternativ PAM sekvenser, ytterligere utvide omfanget av genomisk mål tilgjengelig for manipulasjon18,19,20.
Selv om flere CRISPR-Cas-systemer finnes genomet engineering formål, har de fleste brukere av teknologi stolt på Cas9 nuclease fra Streptococcus pyogenes (SpCas9) av flere grunner. Først krever det en relativt enkelt NGG PAM, i motsetning til mange andre sertifiseringsinstanser proteiner som kan bare holde seg i nærvær av mer komplekse PAMs. Det andre er den første Cas endonuclease å bli distribuert i menneskeceller21,22,23,24. Tredje er SpCas9 langt den beste preget enzymet hittil. Hvis en forsker ønsker å bruke en annen Cas nuclease, vil han eller hun ofte være uklart om hvordan best å utforme eksperimentet og hvor godt andre enzymer vil opptre i ulike biologiske sammenhenger i forhold til SpCas9.
For å utdype den relative ytelsen til forskjellige CRISPR-Cas-systemer, har vi nylig utførte en systematisk Sammenlikning av fem Cas endonucleases-SpCas9, Cas9 enzymet fra Staphylococcus aureus (SaCas9), Cas9 enzymet fra Neisseria meningitidis (NmCas9), Cas12a enzymet fra Acidaminococcus sp. BV3L6 (AsCas12a) og Cas12a enzymet fra Lachnospiraceae bakterie ND2006 (LbCas12a)25. For en rettferdig sammenligning vurdert vi de ulike Cas nucleases bruker samme sett med målområder og andre eksperimentelle forhold. Studien også avgrenset design parameterne for hver CRISPR-Cas-system, som ville tjene som en nyttig referanse for brukere av teknologi. Her, bedre aktiverer forskere å bruke CRISPR-Cas system, gir vi en trinnvis protokoll for optimal genomet engineering med forskjellige Cas9 og Cas12a enzymer (se figur 1). Protokollen inkluderer ikke bare eksperimentelle detaljer men også viktige Utformingshensyn å maksimere sannsynligheten for et vellykket genomet engineering resultat i pattedyrceller.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59086/59086fig1.jpg"> Figur 1 : En oversikt over arbeidsflyten skal generere genomet endret humane cellelinjer. <a href="https://www.jove.com/files/ftp_upload/59086/59086fig1large.jpg" target="_blank">Klikk her for å se en større versjon av dette tallet.</a>

<table border="0" cellpadding="0" cellspacing="0" style="width:1424px;" width="1424">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">T4 Polynucleotide Kinase (PNK)</td>
			<td style="width:175px;">NEB</td>
			<td style="width:119px;">M0201</td>
			<td style="width:688px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Shrimp Alkaline Phosphatase (rSAP)</td>
			<td>NEB</td>
			<td style="width:119px;">M0371</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Tris-Acetate-EDTA (TAE) Buffer, 50X</td>
			<td>1st Base</td>
			<td>BUF-3000-50X4L</td>
			<td>Dilute to 1X before use. The 1X solution contains 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Tris-EDTA (TE) Buffer, 10X</td>
			<td>1st Base</td>
			<td>BUF-3020-10X4L</td>
			<td>Dilute to 1X before use. The 1X solution contains 10 mM Tris (pH 8.0) and 1 mM EDTA.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">BbsI</td>
			<td>NEB</td>
			<td>R0539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">BsmBI</td>
			<td>NEB</td>
			<td>R0580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">T4 DNA Ligase</td>
			<td>NEB</td>
			<td style="width:119px;">M0202</td>
			<td>400,000 units/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Quick Ligation Kit</td>
			<td>NEB</td>
			<td style="width:119px;">M2200</td>
			<td>An alternative to T4 DNA Ligase.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Rapid DNA Ligation Kit</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">K1423</td>
			<td>An alternative to T4 DNA Ligase.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Zero Blunt TOPO PCR Cloning Kit</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">451245</td>
			<td>The salt solution comes with the TOPO vector in the kit.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">NEBuilder HiFi DNA Assembly Master Mix</td>
			<td>NEB</td>
			<td style="width:119px;">E2621L</td>
			<td>Kit for Gibson assembly.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">One Shot Stbl3 Chemically Competent E.Coli</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">C737303</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:443px;">LB Broth (Lennox), powder</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">L3022</td>
			<td>Reconstitute in ddH20, and autoclave before use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:443px;">LB Broth with Agar (Lennox), powder</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">L2897</td>
			<td>Reconstitute in ddH20, and autoclave before use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:443px;">SOC media</td>
			<td>-</td>
			<td style="width:119px;">-</td>
			<td>2.5 mM KCl, 10 mM MgCl2, 20 mM glucose in 1 L of LB Broth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Ampicillin (Sodium), USP Grade</td>
			<td>Gold Biotechnology</td>
			<td style="width:119px;">A-301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">REDiant 2X PCR Mastermix</td>
			<td>1st Base</td>
			<td style="width:119px;">BIO-5185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Agarose</td>
			<td>1st Base</td>
			<td style="width:119px;">BIO-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">T7 Endonuclease I</td>
			<td>NEB</td>
			<td style="width:119px;">M0302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Plasmid DNA Extraction Miniprep Kit</td>
			<td>Favorgen</td>
			<td style="width:119px;">FAPDE 300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Dulbecco&#39;s Modified Eagle Medium (DMEM), High Glucose</td>
			<td>Hyclone</td>
			<td>SH30081.01</td>
			<td>4.5 g/L Glucose, no L-glutamine, HEPES and Sodium Pyruvate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">L-Glutamine, 200mM</td>
			<td>Gibco</td>
			<td style="width:119px;">25030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Penicillin-Streptomycin, 10, 000U/mL</td>
			<td>Gibco</td>
			<td style="width:119px;">15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">0.25% Trypsin-EDTA, 1X</td>
			<td>Gibco</td>
			<td style="width:119px;">25200</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:443px;">Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td style="width:119px;">SV30160</td>
			<td>FBS is heat inactivated before use at 56 oC for 30 min</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Phosphate Buffered Saline, 1X</td>
			<td>Gibco</td>
			<td style="width:119px;">20012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">jetPRIME transfection reagent</td>
			<td>Polyplus Transfection</td>
			<td style="width:119px;">114-75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">QuickExtract DNA Extraction Solution, 1.0</td>
			<td>Epicentre</td>
			<td style="width:119px;">LUCG-QE09050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">ISOLATE II Genomic DNA Kit</td>
			<td>Bioline</td>
			<td style="width:119px;">BIO-52067</td>
			<td>An alternative to QuickExtract</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Q5 High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td style="width:119px;">M0491</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Deoxynucleotide (dNTP) Solution Mix</td>
			<td>NEB</td>
			<td style="width:119px;">N0447</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">6X DNA Loading Dye</td>
			<td>Thermo Scientific</td>
			<td>R0611</td>
			<td>10 mM Tris-HCl (pH 7.6) 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol, 60 mM EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Protease Inhibitor Cocktail, Set3</td>
			<td>Merck</td>
			<td style="width:119px;">539134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Nitrocellulose membrane, 0.2&#181;m</td>
			<td>Bio-Rad</td>
			<td style="width:119px;">1620112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Tris-glycine-SDS buffer, 10X</td>
			<td>Bio-Rad</td>
			<td style="width:119px;">1610772</td>
			<td>Dilute to 1X before use. The 1x solution contains 25 mM Tris, 192 mM glycine, and 0.1% SDS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Tris-glycine buffer, 10X</td>
			<td>1st base</td>
			<td style="width:119px;">BUF-2020</td>
			<td>Dilute to 1X before use. The 1x solution contains 25 mM Tris and 192 mM glycine.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Ponceau S solution</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">P7170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">TBS, 20X</td>
			<td>1st base</td>
			<td style="width:119px;">BUF-3030</td>
			<td>Dilute to 1X before use. The 1x solution contains 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Tween 20</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">P9416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Skim Milk for immunoassay</td>
			<td>Nacalai Tesque</td>
			<td style="width:119px;">31149-75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">WesternBright Sirius-femtogram HRP</td>
			<td>Advansta</td>
			<td style="width:119px;">K12043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Antibody for &#946;-actin (C4)</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-47778</td>
			<td>Lot number: C0916</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">MiSeq system</td>
			<td>Illumina</td>
			<td style="width:119px;">SY-410-1003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">NanoDrop spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">ND-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Qubit fluorometer</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">Q33226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">EVOS FL Cell Imaging System</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">AMF4300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: pSpCas9(BB)-2A-GFP (PX458)</td>
			<td>Addgene</td>
			<td style="width:119px;">48138</td>
			<td>Single vector system: The gRNA is expressed from the same plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA</td>
			<td>Addgene</td>
			<td style="width:119px;">61591</td>
			<td>Single vector system: The gRNA is expressed from the same plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: xCas9 3.7</td>
			<td>Addgene</td>
			<td style="width:119px;">108379</td>
			<td>Dual vector system: The gRNA is expressed from a different plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: pX330-U6-Chimeric_BB-CBh-hSpCas9</td>
			<td>Addgene</td>
			<td style="width:119px;">42230</td>
			<td>Single vector system: The gRNA is expressed from the same plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: hCas9</td>
			<td>Addgene</td>
			<td style="width:119px;">41815</td>
			<td>Dual vector system: The gRNA is expressed from a different plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: eSpCas9(1.1)</td>
			<td>Addgene</td>
			<td style="width:119px;">71814</td>
			<td>Single vector system: The gRNA is expressed from the same plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: VP12 (SpCas9-HF1)</td>
			<td>Addgene</td>
			<td style="width:119px;">72247</td>
			<td>Dual vector system: The gRNA is expressed from a different plasmid.</td>
		</tr>
	</tbody>
</table>

genome editing in mammalian cell lines using crispr-cas

Gruppert regelmessig interspaced kort palindromic gjentar (CRISPR) systemet fungerer naturlig bakteriell adaptiv immunitet, men har blitt vellykket repurposed genomet Engineering i mange forskjellige levende organismer. Oftest wildtype CRISPR tilknyttet 9 (Cas9) eller Cas12a endonuclease brukes til å holde seg bestemte nettsteder i genomet, hvoretter DNA double-strandet pause repareres via ikke-homologe slutten å bli (NHEJ) veien eller homologi-rettet reparasjon ( HDR) sti avhengig av om en giver mal er fraværende eller presentere henholdsvis. Hittil har CRISPR systemer fra ulike bakterie-art vist å kunne utføre genomet redigering i pattedyrceller. Men til tross for den tilsynelatende enkelheten av teknologi må flere design parametere vurderes, som ofte lar brukerne rådvill om hvordan best for å utføre sine genom redigering eksperimenter. Her beskriver vi en komplett arbeidsflyt fra experimental design til identifikasjon av cellen kloner som bærer DNA endringer, med mål om å tilrettelegge vellykket gjennomføring av genomet redigering eksperimenter i pattedyr linjer. Vi markere nøkkelfaktorer for brukere å merke, inkludert valg av CRISPR system, hvor mellomrom og utformingen av en enkelt-strandet oligodeoxynucleotide (ssODN) giver mal. Vi forventer at denne arbeidsflyten vil være nyttig for genet knockout studier, sykdom modellering innsats, eller generering av reporteren cellelinjer.

Utføre et genom redigering eksperiment, en CRISPR plasmider uttrykke en sgRNA rettet mot den locus steder skal klones. Først er plasmider fordøyd med en begrensning enzym (vanligvis en type IIs enzym) å linearize den. Det anbefales å løse fordøyd produktet på en 1% agarose gel sammen med en ufordøyd plasmider å skille mellom en fullstendig og delvis fordøyelsen. Som ufordøyd plasmider er supercoiled, pleier de å kjøre raskere enn sine linearisert kolleger (se figur 7en<...

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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Genomet redigering i pattedyr linjer med CRISPR-Cas

CRISPR-Cas er en kraftig teknologi til ingeniør komplekse genomer av planter og dyr. Her, detalj vi en protokoll effektivt redigere det menneskelige genomet bruker forskjellige Cas-endonucleases. Vi markere viktige hensyn og design parametere for å optimalisere redigering effektivitet.

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

Denne protokollen bruker CRISPR-Cas9 til å opprette knock-out- eller knock-in-linjer. Den største fordelen med denne teknikken er at det er enkelt og effektivt å følge spesielt for nybegynnere forskere. For cellepassaging, behandle overnatten kultiverte celler med to milliliter på 0,25% trypsin EDTA per plate ved 37 grader Celsius i to minutter.Når cellene har løftet fra platebunnen, nøytraliser den enzymatiske reaksjonen med to milliliter cellekulturmedium, og overfør cellesuspensjonen til et konisk rør. Samle cellene ved sentrifugering og gjenbruk pellet i fem milliliter av fersk celle kultur medium for telling. Deretter frø 1,8 ganger 10 til de femte cellene i en brønn av en 24-brønns vev kultur plate for overnatting kultur på 37 grader Celsius og 5% karbondioksid.For transfection av cellene, legg til et passende volum av transfection blanding som inneholder riktig konsentrasjon av CRISPR plasmid til riktig volum av transfection reagens, i henhold til eksperimentell design og produsentens instruksjoner. Etter inkubering av transfection blandingen ved romtemperatur for den anbefalte tidsperioden, legg til løsningen til cellene på en drop-wise måte. Virvle deretter forsiktig platen for å blande og plassere platen i en fuktet 37 grader Celsius inkubator med 5% karbondioksid.På slutten av transfection, tilsett 150 mikroliter trypsin-EDTA å dissosiere cellene som nettopp demonstrert og samle de frittliggende cellene ved sentrifugering. Resuspend pellet i 2% fosterbovin serum i PBS og filtrere cellene gjennom en 30 mikrometer mesh sil i en fem milliliter fluorescens-aktivert celle sortering, eller FACS tube. Bruk deretter de ikke-trans infiserte cellene til å sette en negativ kontrollport på strømningssytometeret og sortere de trans infiserte cellene i henhold til den fluorescerende markøren på CRISPR plasmid som brukes til transinfeksjonen i et rør som inneholder 100 mikroliter cellekulturmedium.Når alle de trans infiserte cellene er samlet inn, pellet cellene ved sentrifugering med maksimal hastighet og resuspend pellet i 300 mikroliter av cellekultur medium. Seed 200 mikroliter av celler i en brønn av en 24-brønns vevskulturplate og la cellene gjenopprette i noen dager i en 37 graders Celsius inkubator. Pellet de resterende 100 mikroliter av sorterte celler med maksimal hastighet i fem minutter og trekke ut genomisk DNA fra den resulterende pellet i henhold til standard DNA ekstraksjon protokoller.Deretter legger du til 10 mikroliter polymerasekjedereaksjonsbuffer, en mikroliter deoksynucleotidblanding, 2,5 mikroliter av brukerdefinert omvendt primer, 0,5 mikroliter DNA-polymerase, to til fem mikroliter av den ekstraherte genomiske DNA-malen og nok dobbeltdestillert vann for å bringe reaksjonen til et endelig volum på 50 mikroliter. Kjør blandingen på en termisk cycler og løse reaksjonen på en 2% agarose gel ved hjelp av 1X TAE buffer i henhold til standardprotokoller. Bruk en ren, skarp skalpell for å avgiftsdirektoratet polymerasekjeden reagere produktet og rense DNA ved hjelp av en gel ekstraksjon kit i henhold til produsentens instruksjoner.Mål konsentrasjonen av produktet ved hjelp av et spektrofotometer ved 260 nanometer absorbans. Forbered en analyseblanding som inneholder 200 nanogram av det isolerte DNA- et, to mikroliter T7-endonukle i reaksjonsbuffer, og nok dobbeltdestillert vann til å bringe det endelige volumet av blandingen til 19 mikroliter. Reanneal polymerase kjedereaksjonsproduktet i en termisk cycler ved de angitte parametrene og bland fem enheter T7 endonukleær I med det reannealed produktet i en 50 minutters inkubasjon ved 37 grader Celsius.På slutten av inkubasjonen, løse fordøyd DNA på en 2,5% agarose gel ved hjelp av 1X TAE buffer, og bilde gelen på en passende gel imaging system. Åpne gelbildet i ImageJ og tegn en rektangulær boks rundt båndet så nær grensen som mulig. Klikk Analyser og Angi målinger, som bekrefter at området, gjennomsnittlig grå verdi og alternativer for integrert tetthet er merket.Klikk OK, og velg Analyser og Mål. Gjennomsnittlig, eller rå intensitetstetthetsverdi, indikerer båndintensiteten. Når de kulturtrans infiserte cellene begynner å bli samløpet, løsne dem med trypsin-EDTA, som demonstrert, og frø cellene sparsomt i en 100 millimeter vevkulturrett for å gi tilstrekkelig plass til individuelle kolonier å vokse før de returnerer cellene til cellekulturinkubatoren.Når koloniene begynner å danne seg, bruk et mikroskop med en fire X forstørrelse for å plukke de enkelte koloniene for overføring til individuelle brønner av en 24-brønnsplate som inneholder 500 mikroliter cellekulturmedium per brønn. Vær forsiktig med at spissen ikke berører de omkringliggende koloniene for å forhindre blanding av kolonier i individuelle brønner eller plukke fra et område med sparsom kolonivekst. Når alle klonene er plukket, plasser platen i cellekulturinkubatoren til kulturene blir samløpet.Som ufordøyde plasmider er super-kveilet, har de en tendens til å kjøre raskere enn sine lineære kolleger. For å finne ut om oligonucleoides har blitt klonet inn i CRISPR plasmid ryggraden, koloni PCR utføres og de positive klonene er inokulert før plasmidene ekstraheres og sendes til Sanger sekvensering. Det fluorescerende signalet kan lett visualiseres under et mikroskop ved vellykket levering av plasmidene, slik at de trans infiserte cellene kan sorteres etter strømningscytometri.En T7 endonukleær I analyse utføres for å sjekke spaltingseffektiviteten til genomisk DNA, som beregnet ut fra intensitetene til båndene observert på en agarosegel. I tillegg, hvis et homologi-rettet reparasjonsbasert eksperiment er utformet for å innlemme et restriksjonssted på målområdet, kan en begrensning fragmentlengde polymorfismeanalyse utføres med et tilsvarende begrensningsenzym. For ytterligere å validere at et proteinkodingsgen har blitt inaktivert, kan en vestlig flekk utføres for å sikre at det ikke finnes noe målrettet protein.Sortering av cellene etter transfeksjon eliminerer cellene uten inkorporerte plasmider, og øker dermed prosentandelen av celler screenet på senere stadier som å ha et knock-out eller knock-in gen. Med utviklingen av denne teknikken er vi nå i stand til å produsere knock-out cellelinjer for å studere genfunksjon og knock-in cellelinjer for å modellere spesifikke sykdommer for å forstå deres mekanisme.

genome-editing-in-mammalian-cell-lines-using-crispr-cas

Research

JoVE Journal

Genetics

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

Genome-wide Fastsettelse av pattedyr Replication Timing av DNA innhold Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Kartlegging av RNA-RNA-interaksjoner globalt ved bruk av biotinylert psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

Kromatinimmunpresipitasjon (ChIP) i mus-T-cellelinjer

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Forbedret genomet redigering med Cas9 Ribonucleoprotein i ulike celler og organismer

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these enhancers combine to produce a transcriptional response. As millions of enhancers have been identified, high-throughput tools are needed to determine enhancer function on a genome-wide scale. Current methods for studying enhancer function include making genetic deletions using nuclease-proficient Cas9, but it is difficult to study the combinatorial effects of multiple enhancers using this technique, as multiple successive clonal cell lines must be generated. Here, we present Enhancer-i, a CRISPR interference-based method that allows for functional interrogation of multiple enhancers simultaneously at their endogenous loci. Enhancer-i makes use of two repressive domains fused to nuclease-deficient Cas9, SID and KRAB, to achieve enhancer deactivation via histone deacetylation at targeted loci. This protocol utilizes transient transfection of guide RNAs to enable transient inactivation of targeted regions and is particularly effective at blocking inducible transcriptional responses to stimuli in tissue culture settings. Enhancer-i is highly specific both in its genomic targeting and its effects on global gene expression. Results obtained from this protocol help to understand whether an enhancer is contributing to gene expression, the magnitude of the contribution, and how the contribution is affected by other nearby enhancers.

This protocol describes the steps needed to design and perform multiplexed targeting of enhancers with the deactivating fusion protein SID4X-dCas9-KRAB, also known as enhancer interference (Enhancer-i). This protocol enables the identification of enhancers that regulate gene expression and facilitates the dissection of relationships between enhancers regulating a common target gene.

Disseksjon Enhancer funksjon med Multiplex CRISPR-baserte Enhancer forstyrrelser i linjer

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

CRISPR-mediert genomet redigering av Human Fungal patogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

Transformasjon, Genova redigering og bestemmelse av fenotype av nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

Cell Surface Reseptor identifikasjon ved hjelp av genom-skala CRISPR / Cas9 genetiske skjermer

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

Vi introduserer punktmutasjoner i menneskelige pluripotente stamceller ved hjelp av sømløs genomredigering

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is an easy-to-use genome editor that has been used in model and non-model species. Here we present a protein-based version of this system that was used to introduce a premature stop codon into a mating gene of a non-model filamentous ascomycete fungus.

CRISPR-Cas9-mediert genomredigering i filamentous ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species ...