Name: Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells (Video) | JoVE Journal
Uploaded: 2025-01-31T12:50:00.000+00:00
Duration: 7 min 37 s
Description: Learn genome-wide CRISPR knockout screening in CAR T cells to investigate resistance mechanisms and improve immunotherapy outcomes. This protocol enables unbiased identification of genetic edits that enhance CAR T cell function and persistence.

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5 minutes

This study was partly funded by the Mayo Clinic Center for Individualized Medicine (SSK), Mayo Clinic Comprehensive&#160;Cancer Center (SSK), Mayo Clinic Center for Regenerative Biotherapeutics (SSK), National Institutes of Health K12CA090628 (SSK) and R37CA266344-01 (SSK), Department of Defense grant CA201127 (SSK), Predolin Foundation (SSK), and Minnesota Partnership for Biotechnology and Medical Genomics (SSK). CMS is supported by the Mayo Clinic Graduate School of Biomedical Sciences. CRISPR screen schematic (Figure 1) was created with BioRender.com (Siegler, L. (2022) https://BioRender.com/k71r054).&#160;

Genome-Wide CRISPR Screens in CART Cells to Investigate Therapy Resistance

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<li>Neelapu, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Axicabtagene+ciloleucel+CAR+T-cell+therapy+in+refractory+large+B-cell+lymphoma%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma.</a> N Engl J Med. 377 (26), 2531-2544 (2017).</li>
<li>Locke, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+safety+and+activity+of+axicabtagene+ciloleucel+in+refractory+large+B-cell+lymphoma+%28zuma-1%29%3A+A+single-arm%2C+multicentre%2C+phase+1-2+trial%5BTitle%5D)+AND+%22Lancet+Oncol%22%5BJournal%5D)">Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (zuma-1): A single-arm, multicentre, phase 1-2 trial.</a> Lancet Oncol. 20 (1), 31-42 (2019).</li>
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<li>Cox, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GM-CSF+disruption+in+CART+cells+modulates+T+cell+activation+and+enhances+CART+cell+anti-tumor+activity%5BTitle%5D)+AND+%22Leukemia%22%5BJournal%5D)">GM-CSF disruption in CART cells modulates T cell activation and enhances CART cell anti-tumor activity.</a> Leukemia. 36 (6), 1635-1645 (2022).</li>
<li>Ottaviano, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phase+1+clinical+trial+of+CRISPR-engineered+CAR19+universal+T+cells+for+treatment+of+children+with+refractory+B+cell+leukemia%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">Phase 1 clinical trial of CRISPR-engineered CAR19 universal T cells for treatment of children with refractory B cell leukemia.</a> Sci Transl Med. 14 (668), eabq3010(2022).</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, 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>
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<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>
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<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>Pattanayak, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+profiling+of+off-target+DNA+cleavage+reveals+RNA-programmed+CAS9+nuclease+specificity%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">High-throughput profiling of off-target DNA cleavage reveals RNA-programmed CAS9 nuclease specificity.</a> Nat Biotechnol. 31 (9), 839-843 (2013).</li>
<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>

SSK is an inventor on patents in the field of CAR immunotherapy that are licensed to Novartis (through an agreement between Mayo Clinic, University of Pennsylvania, and Novartis), Humanigen (through Mayo Clinic), Mettaforge (through Mayo Clinic), and MustangBio (through Mayo Clinic), and Chymal therapeutics (through Mayo Clinic). CS, CMR, and SSK are inventors on patents that are licensed to Immix Biopharma. SSK receives research funding from Kite, Gilead, Juno, BMS, Novartis, Humanigen, MorphoSys, Tolero, Sunesis/Viracta, LifEngine Animal Health Laboratories Inc., and Lentigen. SSK has participated in advisory meetings with Kite/Gilead, Calibr, Luminary Therapeutics, Humanigen, Juno/BMS, Capstan Bio, and Novartis. SSK has served on the data safety and monitoring board with Humanigen and Carisma. SSK has severed a consultant for Torque, Calibr, Novartis, Capstan Bio, BMS, Carisma, and Humanigen. CMS and SSK are inventors of intellectual property that resulted from this protocol.

Gene editing has become a powerful tool in both understanding the mechanisms of resistance to therapies as well as designing novel CART cell therapies to improve the longevity and activity of CART cells16,17,26. While some gene editing strategies have shown improvements in CART cell activity in both preclinical models and clinical trials, there is still work to be done to optimize gene editing strategies. To address this need, r...

Chimeric antigen receptor T (CART) cell therapy has shown impressive success in the treatment of B-cell malignancies; however, the durable response is limited to 30-40%1,2,3,4,5. While researchers have developed and tested several approaches to address mechanisms of resistance to CART cell therapy, including the optimization of CAR design, gene editing, and combination therapies, the development of resistance remains largely unknown. Recently, there has been increasing evidence that the baseline gene expression profile of preinfusion CART cell products is an important determinant of both the toxicity and efficacy of CART therapy6,7,8,9. As such, gene editing during CART cell production has become a popular approach to improve CART cell therapy success.
For instance, our lab previously showed that using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 technology to knock out the gene granulocyte-macrophage colony-stimulating factor (GM-CSF) improves CART cell activity and reduces signs of CART-associated toxicity10,11,12. Additionally, CRISPR-engineered cell therapy has been tested in clinical trials and has shown efficacy and safety13. Together, this indicates that gene editing with CRISPR technology can not only enhance our understanding of CART cell biology but also generate translatable CART cell products.
Genome-wide CRISPR knockout screens have become increasingly popular tools in cancer biology research to understand mechanisms of resistance to therapeutics. In this technique, tens of thousands of guide RNAs (gRNAs) are delivered to pools of cells to encourage the entry of one gRNA per cell14. Then, cells undergo pressure-inducing conditions where cells transduced with gRNAs targeting essential genes die and cells transduced with gRNAs targeting inhibitory genes survive and proliferate. By using next-generation sequencing, we can determine how gRNA representation changes throughout the CRISPR screen14.
However, the scale and selection time required for genome-wide gene perturbations can be challenging to accomplish in primary cells, like CART cells. As such, groups have utilized targeted CRISPR screens to further understand the mechanisms of therapeutic resistance15,16. Targeted screens are often easier to complete in primary cells because they have smaller libraries that require fewer cells to achieve adequate library representation. While these studies have improved our understanding of mechanisms of resistance to CART cell therapy, targeted screens introduce a bias due to the manual selection of gene targets. This article seeks to describe a method to complete an in vitro genome-wide CRISPR knockout screen in CART cells from healthy donors. As such, this high-throughput approach allows for efficient, unbiased identification of key pathways and genes that can be edited to improve therapeutic responses17,18,19.
In particular, the protocol described in this article is designed to increase the field&#39;s understanding of CART cell exhaustion by completing the genome-wide CRISPR knockout screen with an in vitro model for exhaustion. Exhaustion is a dysfunctional CART cell fate that has been implicated in non-response to CART cell therapy20,21,22. This cellular fate is known to be epigenetically regulated, and it is characterized by a decrease in CART cell proliferation, a decrease in effector cytokine production, and an increase in the expression of inhibitory receptors23. In prior literature, gene editing has been able to prevent the development of exhaustion by either up- or downregulating key genes24,25,26. Given both the decrease in proliferative ability as CART cells become exhausted and the evidence that gene editing can prevent its occurrence, we modeled our in vitro genome-wide CRISPR knockout screen on this cellular fate. However, this protocol could be amended in the future to explore other mechanisms of resistance to CART cell therapy.

<table><tbody><tr><td>293T cells</td><td>ATCC</td><td>CRL-3216</td><td>Cells used for lentivirus production</td></tr><tr><td>Biotin ProteinL Antibody</td><td>GenScript</td><td>M00097</td><td>anti-kappa chain antibody for CAR detection</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 antibiotic</td></tr><tr><td>CD4 Isolation Beads</td><td>Miltenyi Biotec</td><td>130-045-101&amp;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&#039;s modified eagle&#039;s medium (DMEM)</td><td>Corning</td><td>10-017-CV</td><td></td></tr><tr><td>Dulbecco&#039;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>Negative isolation kit</td></tr><tr><td>Endura Electrocompetent Cells&amp;nbsp;</td><td>Biosearch Technologies</td><td>60242-1</td><td>Electrocompetent cells with recovery medium</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 library plasmid</td></tr><tr><td>Gene Pulser II&amp;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&lt;sup&gt;+&lt;/sup&gt; target cells&amp;nbsp;</td></tr><tr><td>Lipofectamine 3000 Transfection Reagent</td><td>ThermoFisher Scientific</td><td>L3000075</td><td>Transfection reagent kit with a transfection reagent (Lipofectamine 3000 Reagent) and a neutral co-lipid reagent (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>Density gradient 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>Reduced serum medium</td></tr><tr><td>pCMVR8.74</td><td>AddGene</td><td>22036</td><td>Lentiviral packaging plasmid</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 envelope expressing plasmid</td></tr><tr><td>Pooled Human AB Serum&amp;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 extraction kit</td></tr><tr><td>Qucik-DNA Midiprep Plus Kit</td><td>Zymo Research</td><td>D4075</td><td>Kit used to isolate gDNA</td></tr><tr><td>RoboSep-S</td><td>STEMCELL Technologies</td><td>21000</td><td>Automated cell separator</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>Density gradient separation tube</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>Secondary antibody used for CAR detection</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 &amp;micro;m</td><td>ThermoFisher Scientific</td><td>567-0020</td><td></td></tr><tr><td>Vacuum Filter Systems, 0.45 &amp;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>Hematopoietic cell medium</td></tr></tbody></table>

performing an in vitro genome-wide crispr knockout screen in chimeric antigen receptor t cells

Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells

Chimeric antigen receptor T (CART) cell therapy is an innovative form of targeted immunotherapy that has revolutionized the treatment of cancer. However, the durable response remains limited. Recent studies have shown that the epigenetic landscape of preinfusion CART cell products can influence response to therapy, and gene editing has been proposed as a solution. However, more work needs to be done to determine the optimal gene editing strategy. Genome-wide CRISPR screens have become popular tools to both investigate mechanisms of resistance and optimize gene editing strategies. Yet their application to primary cells presents many challenges. Here we describe a method to complete a genome-wide CRISPR knockout screen in CART cells from healthy donors. As a proof-of-concept model, we designed this method to investigate the development of exhaustion in CART cells targeting the CD19 antigen. However, we believe that this approach can be used to address a variety of mechanisms of resistance to therapy in different CAR constructs and tumor models.

To interrogate genes and pathways that can be edited to improve CART cell activity in an unbiased manner, we designed an in vitro genome-wide CRISPR knockout screen (Figure 1). This screen has two phases: a CART cell production phase and a selective pressure phase. In the CART cell production phase, at least 110 × 106 T-cells are first isolated from healthy donor PBMCs and activated with CD3+/CD28+ beads. The following day, on Day 1,...

The article describes a protocol for the application of an in vitro model for exhaustion to complete a genome-wide CRISPR knockout screen in healthy donor chimeric antigen receptor T cells.

Chimeric antigen receptor T (CART) cell therapy is an innovative form of targeted immunotherapy that has revolutionized the treatment of cancer. However, ...

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Cancer Research

This video demonstrates a technique to manufacture chimeric antigen receptor (CAR) T cells ex vivo. Human T cells are incubated with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, along with a medium supplemented with interleukin-2 (IL-2), leading to T cell activation and proliferation. Transgenic lentiviral vectors are introduced, allowing the transgenic RNA to integrate into the host genome and express the transgene. This results in the transduced T cells expressing chimeric...

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Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

We developed an optimized CRISPR-Cas9 knockout screening protocol for primary CAR T cells. By reducing gDNA carryover through enzymatic digestion and sgRNA cassette pulldown, this approach minimizes PCR artifacts, ensuring accurate sgRNA detection and robust identification of functional gene targets.

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Learn genome-wide CRISPR knockout screening in CAR T cells to investigate resistance mechanisms and improve immunotherapy outcomes. This protocol enables unbiased identification of genetic edits that enhance CAR T cell function and persistence.

Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells

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