Name: Verbetering van CRISPR-Cas9-schermen in CAR T-cellen: Een verfijnde methode voor bibliotheekvoorbereiding (Video) | JoVE Journal
Uploaded: 2026-01-02T13:20:00.000+00:00
Duration: 8 min 3 s
Description: Ontdek een geoptimaliseerde CRISPR-Cas9-screeningmethode voor primaire menselijke CAR T-cellen. Deze techniek overwint belemmeringen in sgRNA-amplificatie uit genomisch DNA en ondersteunt de identificatie van genetische regulatoren.

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<ol><li>Brudno, J. N., Maus, M. V., Hinrichs, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CAR+T+cells+and+T-cell+therapies+for+cancer%3A+A+translational+science+review%5BTitle%5D)+AND+%22JAMA%22%5BJournal%5D)">CAR T cells and T-cell therapies for cancer: A translational science review.</a> JAMA. 332 (22), 1924-1935 (2024).</li>
<li>Cappell, K. M., Kochenderfer, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+outcomes+following+CAR+T+cell+therapy%3A+What+we+know+so+far%5BTitle%5D)+AND+%22Nat+Rev+Clin+Oncol%22%5BJournal%5D)">Long-term outcomes following CAR T cell therapy: What we know so far.</a> Nat Rev Clin Oncol. 20 (6), 359-371 (2023).</li>
<li>Cui, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+challenges+and+progress+of+CAR-T+cell+therapy+in+the+treatment+of+solid+tumors%5BTitle%5D)+AND+%22Mol+Cell+Biochem%22%5BJournal%5D)">The challenges and progress of CAR-T cell therapy in the treatment of solid tumors.</a> Mol Cell Biochem. 480 (10), 5345-5367 (2025).</li>
<li>Shah, N. N., Fry, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+resistance+to+CAR+T+cell+therapy%5BTitle%5D)+AND+%22Nat+Rev+Clin+Oncol%22%5BJournal%5D)">Mechanisms of resistance to CAR T cell therapy.</a> Nat Rev Clin Oncol. 16 (6), 372-385 (2019).</li>
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<li>Bock, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-content+CRISPR+screening%5BTitle%5D)+AND+%22Nat+Rev+Methods+Primers%22%5BJournal%5D)">High-content CRISPR screening.</a> Nat Rev Methods Primers. 2 (1), 1-23 (2022).</li>
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<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>
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<li>Latham, S., Hughes, E., Budgen, B., Morley, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+the+PCR+by+genomic+DNA%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Inhibition of the PCR by genomic DNA.</a> PLoS One. 18 (4), e0284538(2023).</li>
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<li>Giuffre, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overview+of+the+Agilent+Technologies+SureSelectTM+target+enrichment+system%5BTitle%5D)+AND+%22J+Biomol+Tech%22%5BJournal%5D)">Overview of the Agilent Technologies SureSelectTM target enrichment system.</a> J Biomol Tech. 22 (Suppl), S30(2011).</li>
<li>Chen, R., Im, H., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-exome+enrichment+with+the+Roche+NimbleGen+SeqCap+EZ+Exome+Library+SR+platform%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">Whole-exome enrichment with the Roche NimbleGen SeqCap EZ Exome Library SR platform.</a> Cold Spring Harb Protoc. 2015 (7), 634-641 (2015).</li>
<li>Horlbeck, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Compact+and+highly+active+next-generation+libraries+for+CRISPR-mediated+gene+repression+and+activation%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation.</a> eLife. 5, e19760(2016).</li>
<li>Rodriguez-Marquez, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((density+influences+antitumoral+efficacy+of+BCMA+CAR+T+cells+and+correlates+with+clinical+outcome%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">density influences antitumoral efficacy of BCMA CAR T cells and correlates with clinical outcome.</a> Sci Adv. 8 (39), 514(2022).</li>
<li>Gilbert, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-scale+CRISPR-mediated+control+of+gene+repression+and+activation%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Genome-scale CRISPR-mediated control of gene repression and activation.</a> Cell. 159 (3), 647-661 (2014).</li>
<li>Dash, S., Balasubramaniam, M., Dash, C., Pandhare, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biotin-based+pulldown+assay+to+validate+mRNA+targets+of+cellular+miRNAs%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Biotin-based pulldown assay to validate mRNA targets of cellular miRNAs.</a> J Vis Exp. (136), e57786(2018).</li>
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verbetering van crispr-cas9-schermen in car t-cellen: een verfijnde methode voor bibliotheekvoorbereiding

Improving CRISPR-Cas9 Screens in CAR T Cells: A Refined Method for Library Preparation

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

Chimeric antigen receptor (CAR) T cell therapies have demonstrated remarkable efficacy in several hematological malignancies, yet their success has not ...

improving-crispr-cas9-screens-car-t-cells-refined-method-for-library

Research

JoVE Journal

Immunology and Infection

Immunologie en Infectie

The video outlines a process for creating genetically modified CAR T cells through the CRISPR-Cas9 System. Infecting T cells with CRISPR and CAR lentiviruses results in modifications to the target gene and the synthesis of a chimeric antigen receptor or CAR, ultimately leading to the formation of genetically modified CAR T cells.

Genetically Modifying CAR T Cells Using a CRISPR-Cas9 System

Dit protocol beschrijft in detail de generatie van voetafdruk-vrije geïnduceerde pluripotente stamcellen (iPSCs) van menselijke pancreatic cellen onder feeder-vrije, gevolgd door bewerken met behulp van CRISPR/Cas9 ribonucleoproteins en karakterisering van de gewijzigde eencellige klonen.

Efficiënte generatie en het bewerken van Feeder-vrije IPSCs van menselijke Pancreatic cellen met behulp van de CRISPR-Cas9-systeem

CRISPR-Cas is een krachtige technologie om ingenieur van het complex genoom van planten en dieren. Hier, detail we een protocol bij het menselijk genoom met behulp van verschillende Cas enzym efficiënt te bewerken. We benadrukken belangrijke overwegingen en ontwerpparameters bewerken efficiëntie te optimaliseren.

Genoom bewerken in zoogdieren cellijnen met behulp van CRISPR-Cas

Hier presenteren we een protocol voor het optimaliseren van CRISPR-Cas9 om een hogere specificiteit zonder verlies van aan-target activiteit. We een gerichte evolutie aanpak genoemd Sniper-scherm gebruiken om te zoeken een mutant Cas9 met de gewenste kenmerken. Sniper-Cas9 compatible met afgekapte single-gids RNAs en levering in een ribonucleoprotein formaat, bekende strategieën voor het bereiken van hogere specificiteit.

Met behulp van Sniper-Cas9 te minimaliseren Off-target effecten van CRISPR-Cas9 zonder verlies van On-target activiteit Via gerichte evolutie

Een CRISPR/sgRNA-bibliotheek is toegepast op het ondervragen van eiwit-codeert genen. De haalbaarheid van een bibliotheek van de sgRNA aan het licht brengen van de functie van een scheidingswand CTCF in genregulatie blijft echter onontgonnen. Hier beschrijven we een HOX loci specifieke sgRNA bibliotheek te verhelderen van de functie van CTCF grenzen in HOX loci.

HOX Loci gericht CRISPR/sgRNA bibliotheek Screening identificeren kritische CTCF grenzen

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

Dit manuscript beschrijft een genoom-schaal celgebaseerde screening benadering om extracellulaire receptor-ligand interacties te identificeren.

Celoppervlakreceptoridentificatie met genoomschaal CRISPR/Cas9 genetische schermen

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

Het artikel beschrijft een protocol voor de toepassing van een in vitro model voor uitputting om een genoombrede CRISPR-knockoutscreening te voltooien in gezonde chimere antigeenreceptor-T-cellen van de donor.

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

Ontdek een geoptimaliseerde CRISPR-Cas9-screeningmethode voor primaire menselijke CAR T-cellen. Deze techniek overwint belemmeringen in sgRNA-amplificatie uit genomisch DNA en ondersteunt de identificatie van genetische regulatoren.

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

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