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

<ol>
	<li>Neelapu, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-year+follow-up+supports+curative+potential+of+axicabtagene+ciloleucel+in+refractory+large+B-cell+lymphoma+(zuma-1).">5-year follow-up supports curative potential of axicabtagene ciloleucel in refractory large B-cell lymphoma (zuma-1).</a> Blood. 141 (19), 2307-2315 (2023).</li><li>Neelapu, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axicabtagene+ciloleucel+CAR+T-cell+therapy+in+refractory+large+B-cell+lymphoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+safety+and+activity+of+axicabtagene+ciloleucel+in+refractory+large+B-cell+lymphoma+(zuma-1):+A+single-arm,+multicentre,+phase+1-2+trial.">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><li>Maude, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tisagenlecleucel+in+children+and+young+adults+with+B-cell+lymphoblastic+leukemia.">Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia.</a> N Engl J Med. 378 (5), 439-448 (2018).</li><li>Schuster, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tisagenlecleucel+in+adult+relapsed+or+refractory+diffuse+large+B-cell+lymphoma.">Tisagenlecleucel in adult relapsed or refractory diffuse large B-cell lymphoma.</a> N Engl J Med. 380 (1), 45-56 (2018).</li><li>Bai, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+antigen-specific+landscape+of+CAR+T+infusion+product+identifies+determinants+of+CD19-positive+relapse+in+patients+with+all.">Single-cell antigen-specific landscape of CAR T infusion product identifies determinants of CD19-positive relapse in patients with all.</a> Sci Adv. 8 (23), eabj2820 (2022).</li><li>Fraietta, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+response+and+resistance+to+CD19+chimeric+antigen+receptor+(CAR)+T+cell+therapy+of+chronic+lymphocytic+leukemia.">Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia.</a> Nat Med. 24, 563-571 (2018).</li><li>Gennert, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+chromatin+regulatory+landscape+of+human+CAR+T+cell+exhaustion.">Dynamic chromatin regulatory landscape of human CAR T cell exhaustion.</a> Proc Natl Acad Sci USA. 118 (30), e2104758118 (2021).</li><li>Deng, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+anti-CD19+CAR+T+cell+infusion+products+associated+with+efficacy+and+toxicity+in+patients+with+large+B+cell+lymphomas.">Characteristics of anti-CD19 CAR T cell infusion products associated with efficacy and toxicity in patients with large B cell lymphomas.</a> Nat Med. 26 (12), 1878-1887 (2020).</li><li>Sterner, R. M., Cox, M. J., Sakemura, R., Kenderian, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+CRISPR/CAS9+to+knock+out+GM-CSF+in+CAR-T+cells.">Using CRISPR/CAS9 to knock out GM-CSF in CAR-T cells.</a> J Vis Exp. (149), e59629 (2019).</li><li>Sterner, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM-CSF+inhibition+reduces+cytokine+release+syndrome+and+neuroinflammation+but+enhances+CAR-T+cell+function+in+xenografts.">GM-CSF inhibition reduces cytokine release syndrome and neuroinflammation but enhances CAR-T cell function in xenografts.</a> Blood. 133 (7), 697-709 (2019).</li><li>Cox, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM-CSF+disruption+in+CART+cells+modulates+T+cell+activation+and+enhances+CART+cell+anti-tumor+activity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+1+clinical+trial+of+CRISPR-engineered+CAR19+universal+T+cells+for+treatment+of+children+with+refractory+B+cell+leukemia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+CRISPR-CAS9+knockout+and+transcriptional+activation+screening.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+and+epigenetic+regulators+of+human+CD8++T+cell+function+identified+through+orthogonal+CRISPR+screens.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+activation+screen+to+optimize+chimeric+antigen+receptor+(CAR)+T+cell+immunophenotype.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+screening+of+CAR+T+cells+and+cancer+stem+cells+reveals+critical+dependencies+for+cell-based+therapies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+CRISPR+screens+in+primary+human+T+cells+reveal+key+regulators+of+immune+function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+crispr+screens+of+T+cell+exhaustion+identify+chromatin+remodeling+factors+that+limit+T+cell+persistence.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+ATAC-seq+maps+the+comprehensive+and+dynamic+chromatin+accessibility+landscape+of+CAR-T+cell+dysfunction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preinfusion+polyfunctional+anti-CD19+chimeric+antigen+receptor+T+cells+are+associated+with+clinical+outcomes+in+NHL.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+functional+signatures+associated+with+CAR+T+cell+exhaustion+and+impaired+clinical+response+in+patients+with+B+cell+malignancies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cell+exhaustion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deleting+DNMT3A+in+CAR+T+cells+prevents+exhaustion+and+enhances+antitumor+activity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-jun+overexpression+in+CAR+T+cells+induces+exhaustion+resistance.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+inhibition+via+CRISPR+promotes+the+long-term+efficacy+of+CAR+T+cells+against+solid+tumors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+CRISPR-CAS9+knockout+and+transcriptional+activation+screening.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+control,+modeling,+and+visualization+of+CRISPR+screens+with+MAGeCK-VISPR.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+cancer-associated+fibroblasts+in+the+bone+marrow+prevents+resistance+to+CART-cell+therapy+in+multiple+myeloma.">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. 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Bbmerge Available from: <a target="_blank" href="https://github.com/BioInfoTools/BBMap/blob/master/sh/bbmerge.sh">https://github.com/BioInfoTools/BBMap/blob/master/sh/bbmerge.sh</a> (2019)</li><li>Pattanayak, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+profiling+of+off-target+DNA+cleavage+reveals+RNA-programmed+CAS9+nuclease+specificity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+highly+active+sgRNAs+for+CRISPR-CAS9-mediated+gene+inactivation.">Rational design of highly active sgRNAs for CRISPR-CAS9-mediated gene inactivation.</a> Nat Biotechnol. 32 (12), 1262-1267 (2014).</li><li>Dong, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+immunotherapy+target+discovery+using+genome-scale+in+vivo+CRISPR+screens+in+CD8+T+cells.">Systematic immunotherapy target discovery using genome-scale in vivo CRISPR screens in CD8 T cells.</a> Cell. 178 (5), 1189-1204.e23 (2019).</li><li>Zhou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+CRISPR+screens+in+vivo+map+T+cell+fate+regulomes+in+cancer.">Single-cell CRISPR screens in vivo map T cell fate regulomes in cancer.</a> Nature. 624 (7990), 154-163 (2023).</li><li>Sutra Del Galy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genome-wide+CRISPR+screens+identify+SOCS1+as+intrinsic+checkpoint+of+CD4++TH1+cell+response.">In vivo genome-wide CRISPR screens identify SOCS1 as intrinsic checkpoint of CD4+ TH1 cell response.</a> Sci Immunol. 6 (66), eabe8219 (2021).</li><li>Ramos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukemia-intrinsic+determinants+of+CAR-T+response+revealed+by+iterative+in+vivo+genome-wide+CRISPR+screening.">Leukemia-intrinsic determinants of CAR-T response revealed by iterative in vivo genome-wide CRISPR screening.</a> Nat Commun. 14 (1), 8048 (2023).</li></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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#39;s modified eagle&#39;s medium (DMEM)</td>
			<td>Corning</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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&#160;</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&#160;</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+ target cells&#160;</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&#160;</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 &#181;m</td>
			<td>ThermoFisher Scientific</td>
			<td>567-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Filter Systems, 0.45 &#181;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

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 &#215; 106 T-cells are first isolated from healthy donor PBMCs and activated with CD3+/CD28+ beads. The following day, on Day 1</str...

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

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, ...

performing-an-vitro-genome-wide-crispr-knockout-screen-chimeric

Research

JoVE Journal

Cancer Research

501 Views.  Mayo Clinic. 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. 

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

An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of associated loci. In addition, the presence of specific modifications has been used to determine the position and identity non-coding functional elements such as enhancers. In recent years, chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) has become a powerful tool in determining the genome-wide profiles of individual histone modifications. However, it has become increasingly clear that the combinatorial patterns of chromatin modifications, referred to as Chromatin States, determine the identity and nature of the associated genomic locus. Therefore, workflows consisting of robust high-throughput (HT) methodologies for profiling a number of histone modification marks, as well as computational analyses pipelines capable of handling myriads of ChIP-Seq profiling datasets, are needed for comprehensive determination of epigenomic states in large number of samples. The HT-ChIP-Seq workflow presented here consists of two modules: 1) an experimental protocol for profiling several histone modifications from small amounts of tumor samples and cell lines in a 96-well format; and 2) a computational data analysis pipeline that combines existing tools to compute both individual mark occupancy and combinatorial chromatin state patterns. Together, these two modules facilitate easy processing of hundreds of ChIP-Seq samples in a fast and efficient manner. The workflow presented here is used to derive chromatin state patterns from 6 histone mark profiles in melanoma tumors and cell lines. Overall, we present a comprehensive ChIP-seq workflow that can be applied to dozens of human tumor samples and cancer cell lines to determine epigenomic aberrations in various malignancies.

Here, we describe an optimized high-throughput ChIP-sequencing protocol and computational analyses pipeline for the determination of genome-wide chromatin state patterns from frozen tumor tissues and cell lines.

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSFk/o CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.

Here, we present a protocol to genetically edit CAR-T cells via a CRISPR/Cas9 system.

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B cell malignancies or multiple myeloma (MM). However, numerous obstacles limit the efficacy and prohibit the widespread use of CAR T cell therapies due to poor trafficking and infiltration into tumor sites as well as lack of persistence in vivo. Moreover, life-threatening toxicities, such as cytokine release syndrome or neurotoxicity, are major concerns. Efficient and sensitive imaging and tracking of CAR T cells enables the evaluation of T cell trafficking, expansion, and in vivo characterization and allows the development of strategies to overcome the current limitations of CAR T cell therapy. This paper describes the methodology for incorporating the sodium iodide symporter (NIS) in CAR T cells and for CAR T cell imaging using [18F]tetrafluoroborate-positron emission tomography ([18F]TFB-PET) in preclinical models. The methods described in this protocol can be applied to other CAR constructs and target genes in addition to the ones used for this study.

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

This protocol describes the methodology for non-invasively tracking T cells genetically engineered to express chimeric antigen receptors in vivo with a clinically available platform.

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.

Here, we describe a protocol in which an acute lymphoblastic leukemia patient-derived xenograft model is used as a strategy to assess and monitor CD19-targeted chimeric antigen receptor T cell-associated toxicities.

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...