Name: genome engineering of primary human b cells using crispr/cas9
Uploaded: 2020-11-03T14:00:00
Description: Watch this Scientific Journal Video about Genome Engineering of Primary Human B Cells Using CRISPR/Cas9 at JoVE.com

Dette arbejde blev finansieret af Children's Cancer Research Fund (CCRF) og NIH R01 AI146009 til B.S.M.

Leukapheresis prøver fra raske donorer blev indhentet fra en lokal blodbank. Alle de her beskrevne eksperimenter blev bestemt til at blive fritaget for forskning af Institutional Review Board (IRB) og blev godkendt af Institutional Biosafety Committee (IBC) ved University of Minnesota.
BEMÆRK: Alle forsøg blev udført i overensstemmelse med den universelle forholdsregel for blodbårne patogener, med sterile/aseptiske teknikker og korrekt biosikkerhedsniveau-2-udstyr.
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Præcis genomteknik i primære humane B-celler har været udfordrende indtil for nylig9,10. Vi havde tidligere udgivet protokoller ved hjælp af CRISPR/Cas9 til at konstruere primære menneskelige B-celler9. Her skitserer vi forbedrede protokoller for B-celleisolation, ekspansion og teknik for at give mulighed for effektiv KO af CD19 eller for at banke ind EGFP.
Her demonstrerer vi, at vores ekspansions...

B-celler er en undergruppe af lymfocyt afstamning stammer fra hæmatopoietiske stamceller. De udfører en kritisk rolle i det adaptive humorale immunsystem ved at producere store mængder antistoffer som reaktion på immunudfordringer1. B-celler er også forløbere for hukommelse B-celler og de uhelbredeligt differentierede, langlivede plasmaceller, hvilket giver varig humoral immunitet2. Plasmaceller er især unikke blandt immunceller i deres evne til at producere store mængder af et specifikt antistof, mens de overlever i år eller årtier3. Derudover gør den lette isolation fra perifert blod B-celle afstamning en fremragende kandidat til nye cellebaserede terapier4.
Tidligere er tilfældige integrationsmetoder, såsom dem, der bruger lentivirale vektorer eller en Tornerose transposon, blevet brugt til at konstruere B-celler til transgenelevering og udtryk5,6,7,8. Den ikke-specifikke karakter af disse tilgange gør det imidlertid vanskeligt at studere de biologiske funktioner i et bestemt gen i B-cellerne og indebærer en iboende risiko for insertionel mutagenese og variabel transgenudtryk og/eller lyddæmpning i terapeutiske omgivelser.
CRISPR/Cas9-systemet er et kraftfuldt genomteknisk værktøj, der giver forskere mulighed for præcist at redigere genomet af forskellige celler i mange arter. For nylig har to grupper, herunder vores egne, med succes udviklet metoder til ex vivo ekspansion og målrettet genom engineering af primære menneskelige B-celler9,10. Vi vil beskrive processen med rensning af primære humane B-celler fra en leukaforeseprøve. Derefter vil vi beskrive vores opdaterede protokol for B-celleudvidelse og aktivering af isolerede B-celler. Vi vil derefter beskrive en proces til at slå en klynge af differentiering 19 (CD19), en specifik B-cellereceptor og et kendetegn ved B-celler ud ved elektroporation for at introducere CRISPR/Cas9 mRNA sammen med CD19 sgRNA i aktiverede B-celler.
Cas9 mRNA bliver oversat og binder sig til CD19 sgRNA for at danne et CRISPR/Cas9-sgRNA ribonucleoprotein kompleks (RNP). Derefter får sgRNA i komplekset Cas9 til at skabe dobbeltstrengsbrud (DSB) ved målsekvensen på ekson 2 af genet. Cellerne reparerer DSB ved at "ikke-homologe ende sammenføjninger" ved at introducere eller slette nukleotider, hvilket fører til frameshift mutation og forårsager genet at blive slået ud. Vi vil derefter måle tabet af CD19 ved flow cytometry og analysere indel dannelse ved at spore indeller ved nedbrydning (TIDE) analyse.
Vi vil derefter beskrive processen med at bruge CRISPR/Cas9 sammen med en rekombinant AAV6-vektor (rAAV6, en donorskabelon til homologistyret reparation (HDR)) til at mægle stedspecifik indsættelse af forbedret grønt fluorescerende protein(EGFP) på det adeno-associerede virusintegrationssted 1 (AAVS1) gen. AAVS1-genet er et aktivt locus uden kendte biologiske funktioner og et AAV viralt integrationssted på det menneskelige genom; derfor betragtes det som en "sikker havn" for genomteknik. Her rapporterer vi, at udvidelse og aktivering af B-celler tillod op til 44 gange ekspansion i 7 dages kultur (Figur 1). Elektroporation af B-celler viste en mindre reduktion af den samlede cellesundhed (figur 2A) ved 24 timers post-transinfektion. Analysen af spredningsplottet af CD19-markøren (figur 2B) viste en reduktion på op til 83 % i de redigerede celler (figur 2C).
TIDE-analysen af kromotograferne (figur 3A) viste, at % af indel svarede til % proteintabet ved strømningscytometometri (figur 3B). Flowcytometrianalysen af KI-eksperimentet viste, at de celler, der modtog AAV-vektor (Figur 4), sammen med RNP, udtrykte op til 64% EGFP-positive celler (Figur 5A) og senere viste vellykket integration ved hjælp af polymer krydskædereaktion (PCR) (Figur 5B). Celletællinger viste, at alle prøver hurtigt blev fundet inden for 3 dage efter konstruktionen (Figur 5C).

<table>
	<tbody>
		<tr>
			<td>Alt-R S.p. Cas9 Nuclease V3 protein, 500 ug</td>
			<td>IDT</td>
			<td>1081059</td>
			<td>smaller size is also available</td>
		</tr>
		<tr>
			<td>Amaxa P3 primary cell 4D- Nucleofector X Kit S (32 RCT)</td>
			<td>Lonza</td>
			<td>V4XP-3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium-chloride-potassium (ACK) lysing buffer</td>
			<td>Quality Biological</td>
			<td>118-156-101</td>
			<td></td>
		</tr>
		<tr>
			<td>CleanCap chemically modified Cas9 mRNA</td>
			<td>Trilink Biotechnology</td>
			<td>L-7206-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>CpG ODN 2006 (ODN 7909) 5 mg</td>
			<td>Invivogen</td>
			<td>TLRL-2006-5</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>Cryostor CS10, 100 mL</td>
			<td>STEMCELL Technology</td>
			<td>7930</td>
			<td></td>
		</tr>
		<tr>
			<td>CTS Immune Cell SR</td>
			<td>Thermo Fisher Scientific</td>
			<td>A2596101</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep human B cells isolation kit</td>
			<td>STEMCELL Technology</td>
			<td>17954</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience fixable viability dye eFlour 780</td>
			<td>eBiosciences</td>
			<td>65-0865-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Excellerate B cell media, Xeno-free</td>
			<td>R&#38;D Systems</td>
			<td>CCM031</td>
			<td>B-cell basal medium</td>
		</tr>
		<tr>
			<td>Falcon 14 mL Polypropylene Round-bottom Tube</td>
			<td>Corning</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>R&#38;D Systems</td>
			<td>S11550</td>
			<td>For thawing B cells only</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneMate SnapStrip&#174; 8-Strip 0.2 mL PCR Tubes with Individual Attached Dome Caps</td>
			<td>BioExpress</td>
			<td>T-3035-1 / 490003-692</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone 0.0067M PBS (No Ca, No Mg) or 1x PBS</td>
			<td>GE lifesciences</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza 4D Nucleofector core unit</td>
			<td>Lonza</td>
			<td>AAF-1002B</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza 4D Nucleofector X unit</td>
			<td>Lonza</td>
			<td>AAF-1002X</td>
			<td></td>
		</tr>
		<tr>
			<td>Mega CD40 Ligand</td>
			<td>Enzo Life Sciences</td>
			<td>ALX-522-110-C010</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty</td>
			<td>Sigma-Aldrich</td>
			<td>C1562-1EA</td>
			<td>For freezing cells</td>
		</tr>
		<tr>
			<td>Pen/Strep 100X</td>
			<td>Sigma-Aldrich</td>
			<td>TMS-AB2-C</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP anti-human CD19 clone HIB19</td>
			<td>biolegend</td>
			<td>302228</td>
			<td>smaller size is also available</td>
		</tr>
		<tr>
			<td>rAAV6 SA-GFP pakaging ( with our SA-GFP cassette see Figure 4.)</td>
			<td>Vigene Biosciences</td>
			<td>N/A</td>
			<td>large scale packaging, 1e13 GC/mL, 500 mL</td>
		</tr>
		<tr>
			<td>Recombinant human IL-10 protein 250 ug</td>
			<td>R&#38;D Systems</td>
			<td>217-IL-250</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>Recombinant human IL-15 protein 25 ug</td>
			<td>R&#38;D Systems</td>
			<td>247-ILB-025</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>The Big Easy EasySep Magnet</td>
			<td>STEMCELL Technology</td>
			<td>18001</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>Tris-EDTA (TE) buffer</td>
			<td>Fisher Scientific</td>
			<td>BP2476100</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome engineering of primary human b cells using crispr/cas9

B-celler er lymfocytter stammer fra hæmatopoietiske stamceller og er en vigtig del af den humoristiske arm af adaptive immunsystemet. De gør attraktive kandidater til celle-baserede behandlinger på grund af deres lette isolation fra perifere blod, deres evne til at udvide in vitro, og deres levetid in vivo. Derudover kan deres normale biologiske funktion - til at producere store mængder antistoffer - bruges til at udtrykke meget store mængder af et terapeutisk protein, såsom et rekombinant antistof til bekæmpelse af infektion eller et enzym til behandling af enzymopatier. Her leverer vi detaljerede metoder til isolering af primære humane B-celler fra perifere mononukleare blodceller (PBMCs) og aktivering/udvidelse af isolerede B-celler in vitro. Derefter demonstrerer vi de trin, der er involveret i brugen af CRISPR/Cas9-systemet til stedsspecifik ko af endogene gener i B-celler. Denne metode giver mulighed for effektiv KO af forskellige gener, som kan bruges til at studere de biologiske funktioner af gener af interesse. Derefter demonstrerer vi trinene til brug af CRISPR/Cas9-systemet sammen med en rekombinant, adeno-associeret, viral (rAAV) vektor for effektiv stedsspecifik integration af en transgene udtrykskassette i B-celler. Sammen giver denne protokol en trinvis teknisk platform, der kan bruges i primære menneskelige B-celler til at studere biologiske funktioner af gener såvel som til udvikling af B-celleterapi.

Den opdaterede udvidelses- og aktiveringsprotokol muliggjorde en hurtig udvidelse af B-celler op til 44 gange på 7 dage(figur 1;n =3 donorer). I KO-eksperimentet viste B-celletallet ved hjælp af Trypan blå farvning mere end 80% levedygtige celler med en lille reduktion i cellegenvinding i både kontrol- og CD19 KO-prøverne ved 24 timers post-elektroporation (Figur 2A; p ≥ 0,05, n = 3 donorer). Dette resultat indikerer, at elektroporation påvirkede den sam...

Watch this Scientific Journal Video about Genome Engineering of Primary Human B Cells Using CRISPR/Cas9 at JoVE.com

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

Her leverer vi en detaljeret, trin-for-trin-protokol til CRISPR/Cas9-baseret genomteknik af primære humane B-celler til gen knockout (KO) og knock-in (KI) til undersøgelse af biologiske funktioner af gener i B-celler og udvikling af B-celleterapi.

Genomteknik af primære humane B-celler ved hjælp af CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

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