Name: Migliorare gli schermi CRISPR-Cas9 nelle cellule CAR T: un metodo raffinato per la preparazione delle librerie (Video) | JoVE Journal
Uploaded: 2026-01-02T13:20:00.000+00:00
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Description: Scopri un protocollo CRISPR-Cas9 ottimizzato per cellule CAR T primarie umane. Il metodo utilizza digestione enzimatica e pull-down selettivo per migliorare il recupero degli sgRNA e identificare regolatori genetici chiave.

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<li>Gurusamy, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-phenotype+CRISPR-Cas9+screen+identifies+p38+kinase+as+a+target+for+adoptive+immunotherapies%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">Multi-phenotype CRISPR-Cas9 screen identifies p38 kinase as a target for adoptive immunotherapies.</a> Cancer Cell. 37 (6), 818-833.e9 (2020).</li>
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<li>Dong, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+immunotherapy+target+discovery+using+genome-scale+in+vivo+CRISPR+screens+in+CD8+T+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Systematic immunotherapy target discovery using genome-scale in vivo CRISPR screens in CD8 T cells.</a> Cell. 178 (5), 1189-1204.e23 (2019).</li>
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<li>Ye, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genome-scale+gain-of-function+CRISPR+screen+in+CD8+T+cells+identifies+proline+metabolism+as+a+means+to+enhance+CAR-T+therapy%5BTitle%5D)+AND+%22Cell+Metab%22%5BJournal%5D)">A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy.</a> Cell Metab. 34 (4), 595-614.e14 (2022).</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>Yukl, S. A., Kaiser, P., Kim, P., Li, P., Wong, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advantages+of+using+the+QIAshredder+instead+of+restriction+digestion+to+prepare+DNA+for+droplet+digital+PCR%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">Advantages of using the QIAshredder instead of restriction digestion to prepare DNA for droplet digital PCR.</a> BioTechniques. 56 (4), 194(2014).</li>
<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>
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<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>
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<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>
<li>Tsuji, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+biotinylated+RNA+or+DNA+pulldown+assays+for+detection+of+binding+proteins%3A+Examples+of+IRP1%2C+IRP2%2C+HuR%2C+AUF1%2C+and+Nrf2%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Optimization of biotinylated RNA or DNA pulldown assays for detection of binding proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2.</a> Int J Mol Sci. 24 (4), 3604(2023).</li>
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</ol>

<table><tbody><tr><td>4200 TapeStation</td><td>Agilent</td><td>G2991A</td><td></td></tr><tr><td>Acridine Orange</td><td>Invitrogen</td><td>A1301</td><td></td></tr><tr><td>AMPure XP&nbsp;</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>Separatore NEO autoMACS</td><td>Miltenyi</td><td>130-120-327</td><td>Selezionatore a celle magnetiche ad alta velocità</td></tr><tr><td>Oligo biotinilati</td><td>IDT</td><td></td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A9647</td><td></td></tr><tr><td>Contatore a celle fluorescenti K2 del cellometro</td><td>Revvity</td><td>CMT-K2-MX-150</td><td>Contatore a celle fluorescenti</td></tr><tr><td>Camere di conteggio CHT4</td><td>Revvity</td><td>CHT4-SD100-002</td><td></td></tr><tr><td>Nanoperle CD4 di CytoSinct</td><td>GenScript</td><td>L00863-1</td><td></td></tr><tr><td>Nanosfere CytoSinct CD8</td><td>GenScript</td><td>L00864-1</td><td></td></tr><tr><td>dNTP</td><td>Agilent</td><td>200418-51</td><td></td></tr><tr><td>Dynabeads M-280 Streptavidin</td><td>Invitrogen</td><td>11206D</td><td></td></tr><tr><td>Calamita DynaMag-2</td><td>Invitrogen</td><td>12321D</td><td></td></tr><tr><td>EDTA 0,5 M pH 8,0</td><td>Invitrogen</td><td>15575-038</td><td></td></tr><tr><td>Etanolo</td><td>Merck</td><td>1,00,98,31,000</td><td></td></tr><tr><td>Elettroporatore ExPERT ATX</td><td>MaxCyte</td><td>ExPERT ATx</td><td></td></tr><tr><td>Ficoll-Paque</td><td>Cytiva</td><td>17544203</td><td></td></tr><tr><td>Filtropur S 0.2 & micro; m</td><td>Sarstedt</td><td>83,18,26,001</td><td></td></tr><tr><td>GenCRISPR Ultra NLS-Cas9-Research</td><td>GenScript</td><td>Z03621-1</td><td></td></tr><tr><td>Buffer Herculase 5X</td><td>Agilent</td><td>600675-52</td><td>Buffer 5X</td></tr><tr><td>Enzima di fusione dell'Erculasi II</td><td>Agilent</td><td>600679-51</td><td>DNA polimerasi</td></tr><tr><td>IL-15</td><td>Miltenyi</td><td>130-095-765</td><td></td></tr><tr><td>IL-7</td><td>Miltenyi</td><td>130-095-362</td><td></td></tr><tr><td>Mastercycler X50a</td><td>Eppendorf</td><td>6313HG006059</td><td></td></tr><tr><td>Buffer di Elettroporazione MaxCyte</td><td>Cytiva</td><td>EPB1</td><td></td></tr><tr><td>NdeI</td><td>New England Biolabs</td><td>R0111L</td><td></td></tr><tr><td>Tessuto nucleospin</td><td>Macherey-Nagel</td><td>74,09,52,250</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Polybrene</td><td>Merck</td><td>TR-1003-6</td><td></td></tr><tr><td>Primers</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>Propidio Iodio</td><td>Sigma</td><td>P4864</td><td></td></tr><tr><td>PspXI</td><td>New England Biolabs</td><td>R0656S</td><td></td></tr><tr><td>Puromicina</td><td>Gibco</td><td>A11138-03</td><td></td></tr><tr><td>Fluorometro Qubit 3.0</td><td>Invitrogen</td><td>Q33216</td><td></td></tr><tr><td>Test del DNA Qubit HS</td><td>Invitrogen</td><td>Q32851</td><td></td></tr><tr><td>Assemblaggio di elaborazione a pozzo sterile R-50x3</td><td>MaxCyte</td><td>LM243882</td><td></td></tr><tr><td>T Cell TransAct umano</td><td>Miltenyi</td><td>130-111-160</td><td>Reagente di stimolazione delle cellule T</td></tr><tr><td>Stazione a nastro HSD1000</td><td>Agilent</td><td>5067-5585</td><td></td></tr><tr><td>TEXMACS</td><td>Miltenyi</td><td>130-097-196</td><td>Mezzo di crescita per cellule T</td></tr><tr><td>Tris Buffer, 1,0 M, pH 8,0, grado di biologia molecolare</td><td>EMD Milipore</td><td>648314</td><td></td></tr><tr><td>Acqua, per biologia molecolare, senza DNAsi, RNAsi e proteasi</td><td>ThermoScientific</td><td>327390010</td><td></td></tr></tbody></table>

migliorare gli schermi crispr-cas9 nelle cellule car t: un metodo raffinato per la preparazione delle librerie

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

Migliorare gli schermi CRISPR-Cas9 nelle cellule CAR T: un metodo raffinato per la preparazione delle librerie

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

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JoVE Journal

Immunology and Infection

Immunologia e Infezioni

Il video delinea un processo per la creazione di cellule CAR T geneticamente modificate attraverso il sistema CRISPR-Cas9. L'infezione delle cellule T con lentivirus CRISPR e CAR provoca modifiche al gene bersaglio e la sintesi di un recettore chimerico dell'antigene o CAR, portando infine alla formazione di cellule T CAR geneticamente modificate.

Cellule CAR T geneticamente modificate utilizzando un sistema CRISPR-Cas9

Questo protocollo descrive in dettaglio la generazione di cellule staminali pluripotenti indotte privo di impronta (iPSCs) da cellule pancreatiche umane in condizioni prive di alimentatore, seguito da editing con CRISPR/Cas9 ribonucleoproteine e caratterizzazione del modificati cloni unicellulari.

Efficiente generazione e la modifica di IPSCs privo di alimentatore da cellule pancreatiche umane utilizzando il sistema di CRISPR-Cas9

CRISPR-Cas è una potente tecnologia per progettare i complessi genomi delle piante e degli animali. Qui, dettagliamo un protocollo efficiente modificare il genoma umano usando diverse endonucleasi di Cas. Evidenziamo considerazioni importanti e parametri di progettazione per ottimizzare l'efficienza di editing.

Modifica di genoma in linee cellulari di mammifero usando CRISPR-Cas

Qui, presentiamo un protocollo per ottimizzare CRISPR-Cas9 per ottenere una maggiore specificità senza la perdita di attività sul bersaglio. Utilizziamo un approccio diretto evoluzione chiamato Sniper-schermo per trovare un mutante Cas9 con le caratteristiche desiderate. Cecchino-Cas9 è compatibile con tronco singolo-guida RNAs e consegna in un formato della ribonucleoproteina, ben note strategie per conseguire maggiore specificità.

Utilizzando il cecchino-Cas9 a minimizzare gli effetti di fuori bersaglio di CRISPR-Cas9 senza la perdita di attività On-target tramite Evolution diretto

Una libreria CRISPR/sgRNA è stata applicata a interrogare i geni di proteina-codificazione. Tuttavia, la fattibilità di una libreria di sgRNA per scoprire la funzione di un confine CTCF nella regolazione genica rimane inesplorata. Qui, descriviamo una raccolta di specifiche sgRNA loci HOX per delucidare la funzione dei confini CTCF in loci HOX .

HOX Loci Focused CRISPR/sgRNA biblioteca Screening identificazione critico CTCF confini

La tecnologia CRISPR-Cas9 fornisce un metodo efficiente per modificare con precisione il genoma dei mammiferi in qualsiasi tipo di cellula e rappresenta un nuovo mezzo per eseguire schermi genetici a livello di genoma. Qui viene fornito un protocollo dettagliato che illustra i passaggi necessari per il successo delle prestazioni degli schermi CRISPR-Cas9 a livello di genoma raggruppato.

Schermi genetici basati su CRISPR in cellule mammaliane

Questo manoscritto descrive un approccio di screening basato sulle cellule su scala genomica per identificare le interazioni recettore-ligando extracellulari.

Identificazione del recettore della superficie cellulare utilizzando schermi genetici CRISPR/Cas9 su scala genomica

Qui descriviamo una metodologia di screening CRISPR/Cas9 in vivo rapida e diretta utilizzando iniezioni lentivirali embrionali guidate da ultrasuoni in utero per valutare contemporaneamente le funzioni di diversi geni nella pelle e nella cavità orale dei topi immunocompetenti.

Screening CRISPR/Cas9 in vivo per valutare contemporaneamente la funzione genica nella pelle del topo e nella cavità orale

Qui, presentiamo un protocollo per l'editing genetico nelle cellule T umane primarie utilizzando la tecnologia CRISPR Cas per modificare le cellule CAR-T.

Produzione di cellule CAR-T umane ingegnerizzate CRISPR

L'articolo descrive un protocollo per l'applicazione di un modello in vitro per l'esaurimento per completare uno screening CRISPR knockout per l'intero genoma in cellule T del recettore dell'antigene chimerico del donatore sano.

Esecuzione di uno screening CRISPR knockout in vitro a livello di genoma in cellule T del recettore dell'antigene chimerico

Scopri un protocollo CRISPR-Cas9 ottimizzato per cellule CAR T primarie umane. Il metodo utilizza digestione enzimatica e pull-down selettivo per migliorare il recupero degli sgRNA e identificare regolatori genetici chiave.

Migliorare gli schermi CRISPR-Cas9 nelle cellule CAR T: un metodo raffinato per la preparazione delle librerie

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