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. understøttes af et agentur for videnskab teknologi og forsknings fælles Rådet Office grant (1431AFG103), en National Medical Research Council tildele (OFIRG/0017/2016), Grundforskningsfond tilskud (NRF2013-THE001-046 og NRF2013-THE001-093), en Ministeriet for uddannelse Tier 1 tilskud (RG50/17 (S)), en start indrømmer Nanyang Technological University og midler for den internationale genetisk Engineering maskine (iGEM) konkurrence fra Nanyang Technological University.

1. design af sgRNAs
<ol><li>Vælg et passende CRISPR-Cas-system.<ol>
<li>Først inspicere målområde for PAM sekvenser af alle Cas9 og Cas12a nukleaser, der har vist sig at være funktionelle i pattedyrceller16,21-32. Fem hyppigt anvendte enzymer er givet i tabel 1 sammen med deres respektive PAMs. Bemærk: Udover endonucleases anført i tabel 1, der er andre mindre almindeligt anvendte Cas enzymer, der har været med succes indsat i pattedyrceller samt, såsom en Cas9 n...

CRISPR-Cas-system er en kraftfuld, revolutionerende teknologi til ingeniør genomer og transcriptomes af planter og dyr. Talrige bakteriearter har vist sig for at indeholde CRISPR-Cas systemer, som potentielt kan være tilpasset til genom og transkriptom engineering formål44. Selv om Cas9 liv1975 fra Streptococcus pyogenes (SpCas9) var det første enzym til implementeres med succes i humane celler21,22,<sup class="xref...

Evnen til at ingeniør genomet af alle levende organisme har mange biomedicinske og bioteknologiske applikationer, såsom korrektion af sygdom-forårsager mutationer, opførelsen af præcise cellulære modeller for sygdom undersøgelser eller generation af landbruget afgrøder med ønskværdige egenskaber. Siden århundredeskiftet, forskellige teknologier er udviklet til genom engineering i pattedyrceller, herunder meganucleases1,2,3, zink finger nukleaser4,5, eller transkription aktivere-lignende effektor nukleaser (TALENs)6,7,8,9. Men disse tidligere teknologier er enten svært at programmet eller kedelig at samle, dermed hæmmer deres udbredt vedtagelse i forskningen og industrien.
I de seneste år, de klynger interspaced regelmæssigt korte palindromiske gentagelser (CRISPR) - CRISPR-forbundet (Cas) system er opstået som en kraftfulde nye genom engineering technology10,11. Oprindeligt en adaptive immunsystem i bakterier, det har været succesfuldt implementeret for genom ændring i planter og dyr, herunder mennesker. Den primære årsag, hvorfor CRISPR-Cas har opnået så stor popularitet på så kort tid er der det element, der bringer de vigtigste Cas endonuklease, såsom Cas9 eller Cas12a (også kendt som Cpf1), til den korrekte placering i genomet er blot et kort stykke af kimære enkelt guide RN A (sgRNA), som er ligetil at design og billigt at syntetisere. Efter at blive rekrutteret til mål-webstedet, Cas enzym fungerer som et par af molekylære sakse og kløver det bundne DNA med sin RuvC, HNH eller Nuc domæner12,13,14. Den resulterende dobbelt strandede pause (DSB) er efterfølgende repareres af celler via enten ikke-homologe ende sammenføjning (NHEJ) eller homologi-instrueret reparation (HDR) pathway. I mangel af en reparation skabelon, er DSB repareret af den fejlbehæftede NHEJ vej, der kan give anledning til pseudo-tilfældige indsættelse eller deletion af nukleotider (indels) på webstedet cut, potentielt forårsager frameshift mutationer i gener, protein-kodning. Men i nærværelse af en donor-skabelon, der indeholder de ønskede DNA ændringer, DSB er repareret af high fidelity HDR pathway. Almindelige typer af donor skabeloner omfatter enkeltstrenget oligonukleotider (ssODNs) og plasmider. Førstnævnte bruges typisk, hvis de påtænkte DNA ændringer er små (f.eks. ændring af en enkelt basepar), mens sidstnævnte er typisk bruges hvis man ønsker at indsætte en relativt lang sekvens (f.eks den kodende sekvens af en grøn fluorescerende proteiner eller Normal god landbrugspraksis) i target locus.
Liv1975 aktivitet af Cas protein kræver tilstedeværelsen af et protospacer tilstødende motiv (PAM) på målet site15. PAM Cas9 er ultimo 3' protospacer, mens PAM Cas12a (også kaldet Cpf1) er på 5'-enden i stedet16. Cas-guide RNA komplekse er ude af stand til at indføre en DSB, hvis PAM er fraværende17. Derfor, PAM placerer en begrænsning på de genomisk steder hvor en særlig Cas nukleasen er stand til at kløve. Heldigvis, Cas nukleaser fra forskellige bakteriearter typisk udviser forskellige PAM krav. Derfor, ved at integrere forskellige CRISPR-Cas systemer i vores engineering værktøjskasse, kan vi udvide antallet af websteder, der kan være målrettet i en genom. Desuden kan en naturlig Cas enzym manipuleret eller udviklet sig til at genkende alternative PAM sekvenser, yderligere udvide anvendelsesområdet for genomisk mål tilgængelige for manipulation18,19,20.
Selv om flere CRISPR-Cas systemer er tilgængelige for genom engineering formål, har de fleste brugere af teknologien påberåbt sig hovedsageligt Cas9 nukleasen fra Streptococcus pyogenes (SpCas9) af flere årsager. Først, det kræver en forholdsvis simpelthen NGG PAM i modsætning til mange andre Cas proteiner, der kan kun kløver i overværelse af mere komplekse PAMs. For det andet, det er den første Cas liv1975 implementeres med succes i humane celler21,22,23,24. For det tredje er SpCas9 langt de bedst karakteriserede enzym til dato. Hvis en forsker ønsker at bruge en anden Cas nukleasen, vil han eller hun ofte være uklart, om hvordan man bedst at designe eksperimentet og hvor godt andre enzymer vil udføre i forskellige biologiske sammenhænge i forhold til SpCas9.
For at skabe klarhed for den relative resultater af forskellige CRISPR-Cas systemer, har vi for nylig udført en systematisk sammenligning af 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. Vi vurderet til en fair sammenligning, de forskellige Cas nukleaser bruger det samme sæt af målwebsteder og andre forsøgsbetingelser. Undersøgelsen også afgrænset design parametre for hver CRISPR-Cas-system, som ville tjene som en nyttig reference for brugere af teknologien. Her, for bedre at kunne sætte forskerne til at gøre brug af CRISPR-Cas-system, vi leverer en trinvis protokol for optimal genom engineering med forskellige Cas9 og Cas12a enzymer (Se figur 1). Protokollen ikke kun omfatter eksperimentelle detaljer men også vigtige Designovervejelser at maksimere sandsynligheden for en vellykket genom engineering resultatet i pattedyrsceller.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59086/59086fig1.jpg"> Figur 1 : En oversigt over arbejdsproces til at generere genom redigeret menneskelige cellelinjer. <a href="https://www.jove.com/files/ftp_upload/59086/59086fig1large.jpg" target="_blank">Venligst klik her for at se en større version af dette tal.</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>
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genome editing in mammalian cell lines using crispr-cas

Klyngesystem regelmæssigt interspaced korte palindromiske gentagelser (CRISPR) funktioner naturligt i bakteriel adaptive immunitet, men har været succesfuldt repurposed for genom engineering i mange forskellige levende organismer. Mest almindeligt, vildtype CRISPR forbundet 9 (Cas9) eller Cas12a liv1975 bruges til at kløve specifikke steder i genomet, hvorefter DNA dobbelt-strenget pause er repareret via ikke-homologe slutningen at deltage (NHEJ) vej eller homologi-instrueret reparation ( HDR) pathway afhængigt af om en donor skabelon er fraværende eller præsentere henholdsvis. Til dato, har CRISPR systemer fra forskellige bakteriearter vist sig at være i stand til at udføre genom-redigering i pattedyrsceller. Men trods den tilsyneladende enkelhed af teknologien, flere konstruktionsparametre skal betragtes, som ofte lader brugere forvirret om, hvordan du bedst til at udføre deres genom-redigering eksperimenter. Her beskriver vi en komplet workflow fra eksperimentelle design for identifikation af celle kloner, der bærer ønskede DNA ændringer, med mål at fremme vellykket gennemførelse af genom-redigering eksperimenter i pattedyr cellelinjer. Vi fremhæve vigtige overvejelser for brugerne at tage til efterretning, herunder valg af CRISPR system, spacer længde og udformningen af en enkelt-strenget oligodeoxynucleotide (ssODN) donor skabelon. Vi forestiller os, at denne arbejdsproces vil være nyttig for gen knockout undersøgelser, sygdom modellering indsats, eller generation af reporter cellelinjer.

Til at udføre en genom-redigering eksperiment, en CRISPR plasmid udtrykker en sgRNA målretning locus af interesse skal klones. Først, plasmidet er fordøjet med en restriktion enzym (typisk en type IIs enzym) til at linearize det. Det anbefales at løse fordøjet produktet på en 1%-agarosegel sammen med en ufordøjet plasmid at skelne mellem en fuldstændig og delvis fordøjelsen. Som ufordøjet plasmider er supercoiled, de har tendens til at køre hurtigere end deres lineariseret modparter (Se f...

Watch this Scientific Journal Video about Genome Editing in Mammalian Cell Lines using CRISPR-Cas at JoVE.com

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Genom-redigering i pattedyr cellelinjer ved hjælp af CRISPR-Cas

CRISPR-Cas er en kraftfuld teknologi til ingeniør komplekse genomer af planter og dyr. Vi detalje her, en protokol til effektivt redigere det menneskelige genom ved hjælp af forskellige Cas endonucleases. Vi fremhæve vigtige overvejelser og design parametre til at optimere redigering effektivitet.

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

Denne protokol bruger CRISPR-Cas9 til at oprette knock-out- eller knock-in-linjer. Den største fordel ved denne teknik er, at det er enkelt og effektivt at følge især for nybegyndere forskere. For celle passaging, behandle natten dyrkede celler med to milliliter på 0,25% trypsin EDTA pr. plade ved 37 grader Celsius i to minutter.Når cellerne er løftet fra pladebunden, neutraliseres den enzymatiske reaktion med to milliliter cellekulturmedium, og cellesuspensionen overføres til et konisk rør. Cellerne opsamls ved centrifugering og opstimer pellet i fem milliliter frisk cellekulturmedium til optælling. Så frø 1,8 gange 10 til den femte celler i en brønd af en 24-godt væv kultur plade til natten kultur på 37 grader Celsius og 5% kuldioxid.Ved transinfektion af cellerne tilsættes et passende volumen af transinfektionsblandingen, der indeholder en passende koncentration af CRISPR plasmid, til den passende mængde transinfektionsreagens i overensstemmelse med forsøgsdesignet og fabrikantens anvisninger. Efter inkubering af transfektionsblandingen ved stuetemperatur i den anbefalede periode tilsættes opløsningen til cellerne på en dråbemæssig måde. Derefter forsigtigt hvirvle pladen til at blande og placere pladen i en befugtet 37 grader Celsius inkubator med 5% kuldioxid.Ved afslutningen af transinfektionen tilsættes 150 mikroliter trypsin-EDTA for at adskille cellerne som netop påvist og opsaml de fritliggende celler ved centrifugering. Pelletsuspendrer pellet i 2% føtalt kvægserum i PBS, og cellerne filtreres gennem en 30 mikrometermaskes si i en fem milliliter fluorescensaktiv cellesortering eller FACS-rør. Brug derefter de ikke-transficerede celler til at indstille en negativ kontrolport på flowcytometeret og sortere de transmittede celler i henhold til lysstofrøret på CRISPR plasmid, der anvendes til transfektionen, i et rør, der indeholder 100 mikroliter cellekulturmedium.Når alle de transficerede celler er indsamlet, pellet cellerne ved centrifugering ved maksimal hastighed og resuspend pellet i 300 mikroliter af cellekultur medium. Seed 200 mikroliter af celler i en brønd af en 24-godt væv kultur plade og lad cellerne til at inddrive i et par dage i en 37 grader Celsius inkubator. Pellet de resterende 100 mikroliter af sorterede celler ved maksimal hastighed i fem minutter og udtrække genomisk DNA fra den resulterende pellet i henhold til standard DNA ekstraktionsprotokoller.Dernæst tilsættes 10 mikroliter polymerase kædereaktion buffer, en mikroliter af deoxynucleotid mix, 2,5 mikroliter af brugerdefinerede omvendt primer, 0,5 mikroliter af DNA polymerase, to til fem mikroliter af den udvugningsgenske DNA skabelon og nok dobbeltdestilleret vand til at bringe reaktionen til en endelig mængde 50 mikrolitere. Kør blandingen på en termisk cycler og løse reaktionen på en 2% agarose gel ved hjælp af 1X TAE buffer i henhold til standard protokoller. Brug en ren, skarp skalpel til punktafgifter polymerasekæden reagerer produkt og rense DNA ved hjælp af en gel ekstraktion kit i henhold til producentens anvisninger.Produktets koncentration måles ved hjælp af et spektrofotometer ved 260 nanometerabsor absorbans. Forbered en analyseblanding indeholdende 200 nanogram af det isolerede DNA, to mikroliter T7 endonuclease I-reaktionsbuffer og nok dobbeltdestilleret vand til at bringe det endelige volumen af blandingen op på 19 mikroliter. Reanneal polymerase kædereaktion produkt i en termisk cycler på de angivne parametre og bland fem enheder af T7 endonuclease I med reannealed produkt til en 50 minutters inkubation ved 37 grader Celsius.Ved slutningen af inkubationen skal du fortolke det fordøjede DNA på en 2,5% agarosegel ved hjælp af 1X TAE-buffer og afbilde gelen på et passende gelbilledsystem. Åbn gelbilledet i ImageJ, og tegn en rektangulær boks rundt om båndet så tæt på dets grænse som muligt. Klik på Analysér og Angiv målinger, der bekræfter, at området, den gennemsnitlige grå værdi og de integrerede tæthedsindstillinger er markeret.Klik på OK, og vælg Analysér og Målér. Den gennemsnitlige eller rå intensitetstæthedsværdi er et tegn på båndintensiteten. Når de kultur-transficerede celler begynder at blive sammenløbet, løsrive dem med trypsin-EDTA, som påvist, og frø cellerne sparsomt i en 100 millimeter vævskultur skål for at give tilstrækkelig plads til de enkelte kolonier til at vokse, før de returnerer cellerne til cellekultur inkubator.Når kolonierne begynder at danne, skal du bruge et mikroskop med en fire X forstørrelse til at vælge de enkelte kolonier til overførsel til individuelle brønde af en 24-brønd plade, der indeholder 500 mikroliter cellekultur medium per brønd. Pas på, at dit tip ikke rører de omkringliggende kolonier for at forhindre blanding af kolonier i individuelle brønde eller vælge fra et område med sparsom kolonivækst. Når alle kloner er plukket, skal du placere pladen i cellekulturinkubatoren, indtil kulturerne bliver sammenløbet.Som ufordøjede plasmider er super-oprullet, de har tendens til at køre hurtigere end deres lineariserede kolleger. For at afgøre, om oligonukleotiderne er blevet klonet med succes i CRISPR plasmid rygraden, udføres koloni-PCR, og de positive kloner podes, før plasmiderne udvindes og sendes til Sanger-sekvensering. Fluorescerende signal kan let visualiseres under et mikroskop ved en vellykket levering af plasmider, så de transficerede celler, der skal sorteres efter flow cytometri.En T7 endonuclease I assay udføres for at kontrollere spaltning effektiviteten af det genomiske DNA, som beregnet ud fra intensiteten af de bånd observeret på en agarose gel. Hvis et homologi-styret reparationsbaseret eksperiment desuden er designet til at inkorporere et restriktionssted på målblyolen, kan en begrænsningsfragmentlængde polymorfianalyse udføres med et tilsvarende restriktionsenzym. For yderligere at validere, at et proteinkodningsgen er blevet inaktiveret, kan der udføres en vestlig skamplet for at sikre, at der ikke er noget målrettet protein til stede.Sortering af cellerne efter transfektion eliminerer cellerne uden indarbejdet plasmider, hvilket øger procentdelen af celler screenet på senere stadier som havende en knock-out eller knock-in gen. Med udviklingen af denne teknik, er vi nu i stand til at producere knock-out cellelinjer til at studere genfunktion og knock-in cellelinjer til at modellere specifikke sygdomme for at 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.

Genom-dækkende Bestemmelse af pattedyr Replication Timing ved 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 ...

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.

Kortlægning af RNA-RNA-interaktioner globalt ved anvendelse af biotinyleret 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.

Chromatin-immunpræcipitation (ChIP) i mus-T-cellelinier

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 genom-redigering med Cas9 Ribonucleoprotein i forskellige 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.

Dissektion af Enhancer funktion ved hjælp af Multiplex CRISPR-baserede forstærker interferens i cellelinjer

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-medieret genom-redigering af menneskelige svampe 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.

Omdanne, genomredigering og Phenotyping kvælstofbindende tropisk Cannabaceae træ 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.

Identifikation af cellereceptorer ved hjælp af genome-skala CRISPR/Cas9 genetiske skærme

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).

Introduktion af punktmutationer i humane pluripotente stamceller ved hjælp af problemfri 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-medieret Genom Redigering i filamentøse 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 ...