Name: Uitvoeren van een in vitro genoombrede CRISPR-knock-outscreening in chimere antigeenreceptor-T-cellen (Video) | JoVE Journal
Uploaded: 2025-01-31T12:50:00.000+00:00
Duration: 7 min 37 s
Description: Leer een genoombrede CRISPR-knock-outscreening uitvoeren in CAR-T-cellen om resistentiemechanismen in kankerimmunotherapie te onderzoeken. Deze methode onthult genetische factoren die de therapeutische respons beïnvloeden.

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5 minutes

<meta charset="utf8"></head><body>Genoombrede CRISPR-schermen in CART-cellen om therapieresistentie te onderzoeken

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<li>Joung, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-scale+CRISPR-CAS9+knockout+and+transcriptional+activation+screening%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Genome-scale CRISPR-CAS9 knockout and transcriptional activation screening.</a> Nat Protoc. 12, 828-863 (2017).</li>
<li>Mccutcheon, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+and+epigenetic+regulators+of+human+CD8%2B+T+cell+function+identified+through+orthogonal+CRISPR+screens%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens.</a> Nat Genet. 55, 2211-2223 (2023).</li>
<li>Zhu, W., Kelly, C., Dagur, P., Dunbar, C. E., Cordes, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR+activation+screen+to+optimize+chimeric+antigen+receptor+%28CAR%29+T+cell+immunophenotype%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">CRISPR activation screen to optimize chimeric antigen receptor (CAR) T cell immunophenotype.</a> Blood. 142 (Supplement 1), 4820-4820 (2023).</li>
<li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR+screening+of+CAR+T+cells+and+cancer+stem+cells+reveals+critical+dependencies+for+cell-based+therapies%5BTitle%5D)+AND+%22Cancer+Discov%22%5BJournal%5D)">CRISPR screening of CAR T cells and cancer stem cells reveals critical dependencies for cell-based therapies.</a> Cancer Discov. 11 (5), 1192-1211 (2021).</li>
<li>Shifrut, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+CRISPR+screens+in+primary+human+T+cells+reveal+key+regulators+of+immune+function%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Genome-wide CRISPR screens in primary human T cells reveal key regulators of immune function.</a> Cell. 175 (7), 1958-1971.e15 (2018).</li>
<li>Belk, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+crispr+screens+of+T+cell+exhaustion+identify+chromatin+remodeling+factors+that+limit+T+cell+persistence%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">Genome-wide crispr screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence.</a> Cancer Cell. 40 (7), 768-786.e7 (2022).</li>
<li>Jiang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+ATAC-seq+maps+the+comprehensive+and+dynamic+chromatin+accessibility+landscape+of+CAR-T+cell+dysfunction%5BTitle%5D)+AND+%22Leukemia%22%5BJournal%5D)">Single-cell ATAC-seq maps the comprehensive and dynamic chromatin accessibility landscape of CAR-T cell dysfunction.</a> Leukemia. 36 (11), 2656-2668 (2022).</li>
<li>Rossi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preinfusion+polyfunctional+anti-CD19+chimeric+antigen+receptor+T+cells+are+associated+with+clinical+outcomes+in+NHL%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL.</a> Blood. 132 (8), 804-814 (2018).</li>
<li>Beider, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+and+functional+signatures+associated+with+CAR+T+cell+exhaustion+and+impaired+clinical+response+in+patients+with+B+cell+malignancies%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">Molecular and functional signatures associated with CAR T cell exhaustion and impaired clinical response in patients with B cell malignancies.</a> Cells. 11 (7), 1140(2022).</li>
<li>Wherry, E. J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((cell+exhaustion%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">cell exhaustion.</a> Nat Immunol. 12, 492-499 (2011).</li>
<li>Prinzing, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deleting+DNMT3A+in+CAR+T+cells+prevents+exhaustion+and+enhances+antitumor+activity%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">Deleting DNMT3A in CAR T cells prevents exhaustion and enhances antitumor activity.</a> Sci Transl Med. 13, eabh0272(2021).</li>
<li>Lynn, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((C-jun+overexpression+in+CAR+T+cells+induces+exhaustion+resistance%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">C-jun overexpression in CAR T cells induces exhaustion resistance.</a> Nature. 576, 293-300 (2019).</li>
<li>Tang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TGF-beta+inhibition+via+CRISPR+promotes+the+long-term+efficacy+of+CAR+T+cells+against+solid+tumors%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">TGF-beta inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors.</a> JCI Insight. 5 (4), e133977(2020).</li>
<li>Joung, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-scale+CRISPR-CAS9+knockout+and+transcriptional+activation+screening%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Genome-scale CRISPR-CAS9 knockout and transcriptional activation screening.</a> Nat Protoc. 12 (4), 828-863 (2017).</li>
<li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quality+control%2C+modeling%2C+and+visualization+of+CRISPR+screens+with+MAGeCK-VISPR%5BTitle%5D)+AND+%22Genome+Biol%22%5BJournal%5D)">Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR.</a> Genome Biol. 16, 281(2015).</li>
<li>Sakemura, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+cancer-associated+fibroblasts+in+the+bone+marrow+prevents+resistance+to+CART-cell+therapy+in+multiple+myeloma%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Targeting cancer-associated fibroblasts in the bone marrow prevents resistance to CART-cell therapy in multiple myeloma.</a> Blood. 139 (26), 3708-3721 (2022).</li>
<li>Stewart, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IL-4+drives+exhaustion+of+CD8%2B+CART+cells%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">IL-4 drives exhaustion of CD8+ CART cells.</a> Nat Commun. 15 (1), 7921(2024).</li>
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<li>Doench, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rational+design+of+highly+active+sgRNAs+for+CRISPR-CAS9-mediated+gene+inactivation%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Rational design of highly active sgRNAs for CRISPR-CAS9-mediated gene inactivation.</a> Nat Biotechnol. 32 (12), 1262-1267 (2014).</li>
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<li>Zhou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+CRISPR+screens+in+vivo+map+T+cell+fate+regulomes+in+cancer%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single-cell CRISPR screens in vivo map T cell fate regulomes in cancer.</a> Nature. 624 (7990), 154-163 (2023).</li>
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</ol>

<table><tbody><tr><td>293T cells</td><td>ATCC</td><td>CRL-3216</td><td>Cellen gebruikt voor lentivirus productie</td></tr><tr><td>Biotin ProteinL Antibody</td><td>GenScript</td><td>M00097</td><td>anti-kappa ketting antilichaam voor CAR detectie</td></tr><tr><td>Bovine Serum Albumin</td><td>Millipore Sigma</td><td>A7906</td><td></td></tr><tr><td>Carbenicillin disodium salt</td><td>Millipore Sigma</td><td>C1389-1G</td><td>Carbenicillin antibioticum</td></tr><tr><td>CD4 Isolation Beads</td><td>Miltenyi Biotec</td><td>130-045-101&nbsp;</td><td></td></tr><tr><td>CD8 Isolation Beads</td><td>Miltenyi Biotec</td><td>130-045-201</td><td></td></tr><tr><td>CTS (Cell Therapy Systems) Dynabeads CD3/CD28</td><td>Gibco</td><td>40203D</td><td></td></tr><tr><td>Cytoflex</td><td>Beckman Coulter</td><td>NC2279958</td><td></td></tr><tr><td>DNase-Free Water</td><td>Invitrogen</td><td>AM9937</td><td></td></tr><tr><td>Dulbecco's modified eagle's medium (DMEM)</td><td>Corning</td><td>10-017-CV</td><td></td></tr><tr><td>Dulbecco's Phosphate-Buffered Saline</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>EasySep Human T Cell Isolation Kit</td><td>STEMCELL Technologies</td><td>17951RF</td><td>Negatieve isolatie kit</td></tr><tr><td>Endura Electrocompetent Cells&nbsp;</td><td>Biosearch Technologies</td><td>60242-1</td><td>Electrocompetent cellen met herstelmedium</td></tr><tr><td>Ethanol</td><td>Millipore Sigma</td><td>E7023</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Corning</td><td>35-010-CV</td><td></td></tr><tr><td>GeCKO v2 CRISPR Knockout Pooled Library A</td><td>AddGene</td><td>1000000048</td><td>CRISPR bibliotheek plasmide</td></tr><tr><td>Gene Pulser II&nbsp;</td><td>Bio-Rad</td><td>165-2105</td><td>Electroporator</td></tr><tr><td>Glycogen</td><td>Millipore Sigma</td><td>10901393001</td><td></td></tr><tr><td>JeKo-1</td><td>ATCC</td><td>CRL-3006</td><td>CD19+ doelcellen&nbsp;</td></tr><tr><td>Lipofectamine 3000 Transfection Reagent</td><td>ThermoFisher Scientific</td><td>L3000075</td><td>Transfectiereagens kit met een transfectiereagens (Lipofectamine 3000 Reagens) en een neutraal co-lipide reagens (p3000)</td></tr><tr><td>LIVE/DEAD Aqua</td><td>Invitrogen</td><td>L34966</td><td></td></tr><tr><td>Lymphoprep</td><td>STEMCELL Technologies</td><td>7851</td><td>Dichtheidsgradiënt medium</td></tr><tr><td>Machery-Nagel NucleoBond Xtra Maxi Kits</td><td>ThermoFisher Scientific</td><td>12748412</td><td>Maxi-prep kit</td></tr><tr><td>NEBNext High-Fidelity 2X PCR MasterMix</td><td>New England BioLabs</td><td>M0541S</td><td>High fidelity PCR mastermix</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco</td><td>31985-070</td><td>Medium met verminderd serum</td></tr><tr><td>pCMVR8.74</td><td>AddGene</td><td>22036</td><td>Lentivirale verpakkingsplasmide</td></tr><tr><td>Pennicillin-streptomycin-glutamine (100X)</td><td>Life Technologies</td><td>10378-016</td><td></td></tr><tr><td>pMD2.G</td><td>AddGene</td><td>12259</td><td>VSV-G envelop expresserend plasmide</td></tr><tr><td>Pooled Human AB Serum&nbsp;</td><td>Innovative Research</td><td>ISERABHI</td><td></td></tr><tr><td>Puromycin</td><td>Millipore Sigma</td><td>P8833</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>Gek extractie kit</td></tr><tr><td>Qucik-DNA Midiprep Plus Kit</td><td>Zymo Research</td><td>D4075</td><td>Kit gebruikt om gDNA te isoleren</td></tr><tr><td>RoboSep-S</td><td>STEMCELL Technologies</td><td>21000</td><td>Geautomatiseerde celafscheider</td></tr><tr><td>Roswell Park Memorial Institute 1640 Medium (RPMI)</td><td>Gibco</td><td>21870092</td><td></td></tr><tr><td>SepMate-50</td><td>STEMCELL Technologies</td><td>85450</td><td>Dichtheidsgradiënt scheidingsbuis</td></tr><tr><td>Sodium Acetate</td><td>Invitrogen</td><td>AM9740</td><td></td></tr><tr><td>Sodium Azide</td><td>Fisher Scientific</td><td>71448-16</td><td></td></tr><tr><td>Streptavidin Antibody (PE)</td><td>BioLegend</td><td>405203</td><td>Secundair antilichaam gebruikt voor CAR detectie</td></tr><tr><td>T100 Thermal Cycler</td><td>Bio-Rad</td><td>1861096</td><td></td></tr><tr><td>Ultracentrifuge (Optima XPN-80)</td><td>BeckmanCoulter</td><td>A99839</td><td></td></tr><tr><td>Vacuum Filter Systems, 0.22 &micro;m</td><td>ThermoFisher Scientific</td><td>567-0020</td><td></td></tr><tr><td>Vacuum Filter Systems, 0.45 &micro;m</td><td>ThermoFisher Scientific</td><td>165-0045</td><td></td></tr><tr><td>X-VIVO 15 Serum-Free Hematopoietic Cell Medium</td><td>Lonza</td><td>04-418Q</td><td>Hematopoietisch celmedium</td></tr></tbody></table>

uitvoeren van een in vitro genoombrede crispr-knock-outscreening in chimere antigeenreceptor-t-cellen

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Crispr Cas9-technologie biedt een efficiënte methode om het genoom van zoogdieren in elk celtype nauwkeurig te bewerken en vertegenwoordigt een nieuw middel om genoom-brede genetische schermen uit te voeren. Hier vindt u een gedetailleerd protocol over de stappen die nodig zijn voor het succesvol uitvoeren van gepoolde CRISPR Cas9 schermen.

Gepoolde CRISPR-gebaseerde genetische schermen in zoogdiercellen

Hier beschrijven we een snelle en directe in vivo CRISPR/Cas9 screeningsmethodologie met behulp van echogeleide in utero embryonale lentivirale injecties om tegelijkertijd functies van verschillende genen in de huid en mondholte van immunocompetente muizen te beoordelen.

In Vivo CRISPR/Cas9-screening om tegelijkertijd de genfunctie in de huid en mondholte van de muis te evalueren

Hier presenteren we een protocol voor genbewerking in primaire menselijke T-cellen met behulp van CRISPR Cas-technologie om CAR-T-cellen te wijzigen.

Productie van menselijke CRISPR-engineered CAR-T-cellen

Dit protocol stroomlijnt de productie van retrovirale vectoren en de transductie van T-cellen bij muizen, waardoor de efficiënte generatie van CAR-T-cellen van muizen wordt vergemakkelijkt.

Efficiënte generatie van muizen chimere antigeenreceptor (CAR)-T-cellen

Dit protocol biedt een gestroomlijnde workflow voor het genereren en testen van menselijke chimere antigeenreceptorregulerende T-cellen (CAR Tregs).

Generatie van menselijke chimere antigeenreceptorregulerende T-cellen

Hier beschrijven we een methode voor het maken van dubbele emulsiedruppeltjes die één T-cel en een kankerdoelcel inkapselen om celdoding op eencellig niveau te onderzoeken. Deze methode maakt dubbele kwantificering mogelijk van zowel cytotoxische moleculen als apoptose van doelcellen binnen een grote populatie T-cellen.

Op druppels gebaseerde cytotoxiciteitstest om chimere antigeenreceptor-T-cellen op eencellig niveau te beoordelen

We ontwikkelden een geoptimaliseerd CRISPR-Cas9 knockout-screeningsprotocol voor primaire CAR T-cellen. Door de gDNA-overdracht via enzymatische vertering en sgRNA-cassette pulldown te verminderen, minimaliseert deze aanpak PCR-artefacten, waardoor nauwkeurige sgRNA-detectie en robuuste identificatie van functionele gendoelen worden gegarandeerd.

Verbetering van CRISPR-Cas9-schermen in CAR T-cellen: Een verfijnde methode voor bibliotheekvoorbereiding

Leer een genoombrede CRISPR-knock-outscreening uitvoeren in CAR-T-cellen om resistentiemechanismen in kankerimmunotherapie te onderzoeken. Deze methode onthult genetische factoren die de therapeutische respons beïnvloeden.

Uitvoeren van een in vitro genoombrede CRISPR-knock-outscreening in chimere antigeenreceptor-T-cellen

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