Name: Improving CRISPR-Cas9 Screens in CAR T Cells: A Refined Method for Library Preparation (Video) | JoVE Journal
Uploaded: 2026-01-02T13:20:00.000+00:00
Duration: 8 min 3 s
Description: Discover an optimized CRISPR-Cas9 screening protocol for primary human CAR T cells using enzymatic digestion and selective pulldown. This method improves sgRNA recovery from genomic DNA to identify genetic regulators enhancing CAR T cell therapies.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This study was supported by the PID2022-137914OB-I00 project financed by MICIU/AEI /10.13039/501100011033 and by FEDER, UE. This study was supported by Instituto de Salud Carlos III (ISCIII) through Red de Terapias Avanzadas TERAV and TERAV+ (RD21/0017/0009 and RD24/0014/0010) and through the Centro de Investigacion Biomedica en Red de Cancer CIBERONC (CB16/12/00489). This study was supported by Gobierno de Navarra Salud (GN2023/08 and GN2024/04) and Proyectos Estrat&#233;gicos (DIAMANTE 0011-1411-2023-000105 and 0011-1411-2023-000074). Figure 1B and Figure 2A were created with BioRender.com

This protocol was performed in accordance with Universidad de Navarra guidelines. All subjects provided written informed consent. The reagents and the equipment used in this study are listed in the Table of Materials.
1. T cell isolation and activation
<ol>
	<li>PBMC isolation
	<ol>
		<li>Collect blood samples from donors on ethylenediaminetetraacetic acid (EDTA) tubes, transfer blood samples to 50 mL conical tubes, and dilute blood with PBS (dilution 1:1).</li>
		<li>Add 15 mL of density gradient medium to the bottom of a 50 mL conical tube (density for PBMCs: 1.07). Then, add carefully 25 mL of diluted blood over the density gradient medium layer (slowest speed of the pipette controller). Centrifuge for 30 min at 800 &#215; g without brake.</li>
		<li>Collect PBMCs ring/layer with a Pasteur pipette into a 50 mL conical tube and add PBS to a total volume of 50 mL to wash. Centrifuge at 650 &#215; g for 8 min. Discard the supernatant. 
		NOTE: Check the turbidity of the supernatant to be sure that there are no cells there. If in doubt, divide the suspension into twice the original number of 50 mL conical tubes, add PBS until 50 mL, and centrifuge again.</li>
		<li>Resuspend in 50 mL of PBS. Count with acridine orange-propidium iodide (AOPI) in a cell counter. Centrifuge at 650 &#215; g for 8 min.</li>
	</ol>
	</li>
	<li>CD4 and CD8 magnetic selection
	<ol>
		<li>Prepare fluorescence-activated cell sorting (FACS) buffer: PBS, EDTA 2.5 &#181;M, BSA 0.5% and filter through 0.2 &#181;m.</li>
		<li>Resuspend PBMCs in 80 &#181;L of FACS buffer sterile per 10 &#215; 106 cells and add 10 &#181;L of microbeads CD4 and 10 &#181;L of microbeads CD8 per 10 &#215; 106 cells. Incubate 20 min at 4 &#176;C.</li>
		<li>Add 10 mL of FACS buffer and centrifuge for 8 min at 650 &#215; g. Discard the supernatant completely.</li>
		<li>Resuspend in 500 &#181;L per 1 &#215; 108 cells of FACS Buffer in a 15 mL conical tube. 
		NOTE: A run can accommodate 200 x 106 cells.</li>
		<li>Use the Possel program for AutoMacs (high-speed magnetic cell sorter) isolation.</li>
		<li>Add up to 10 mL of PBS, count with AOPI in a fluorescent cell counter, and centrifuge at 650 &#215; g for 8 min.</li>
		<li>Resuspend cells 1 x 106/mL in T cell growth medium containing 3% human&#160;serum (HS), 1% penicillin/streptomycin (P/S), 625 IU/mL interleukin (IL)-7, 85 IU/mL IL-15. Add 10 &#181;L of T cell stimulation reagent per 1 mL and mix well. Plate 2 mL per well in a 24 well plate (p24w) and incubate for 24 h at 37 &#176;C.</li>
	</ol>
	</li>
</ol>
2. CAR T cell production and CRISPR screening
<ol>
	<li>Library spinfection and CAR transduction
	<ol>
		<li>Add the corresponding amount of lentivirus CRISPR library (e.g., Brunello Kinome 1 library. See Supplementary Table 1), determined in a titration assay, and 8 &#181;g/mL polybrene.</li>
		<li>Spinfect by centrifugation at 700 x g for 1 h 30 min at 32 &#176;C.</li>
		<li>Replace media with fresh T cell growth medium containing 3% HS, 1% P/S, 625 IU/mL IL-7, 85 IU/mL IL-15 (final [cell]= 106 cells/mL).</li>
		<li>After 6 h, add the corresponding amount of CAR lentiviral vector to a multiplicity of infection (MOI) of 3 and incubate for 4 days.</li>
	</ol>
	</li>
	<li>Cas 9 electroporation
	<ol>
		<li>Collect CAR T cells, wash once with PBS, and count cells with AOPI in a fluorescent cell counter.</li>
		<li>Prepare 4 &#181;M Cas9 in electroporation buffer.</li>
		<li>Resuspend CAR T cells in 50 &#181;L per 5 &#215; 106&#160;cells. Mix and carefully add 50 &#181;L of cell suspension in each well of a multiwell cuvette strip without forming bubbles. Then, using a pipette tip, remove any possible bubbles and run the tip along the edges of the well. 
		NOTE: If any well in a cuvette is left without a sample, add the same volume of electroporation Buffer. This is a critical step: if the machine detects a bubble or volume difference, it will give an error.</li>
		<li>Use the program EXPAND T CELL 3 to nucleofect CAR T cells.</li>
		<li>Leave the cells in the cuvettes in an incubator at 37 &#176;C for 40 min.</li>
		<li>Resuspend the nucleofected cells in T cell growth medium containing 3% HS, 1% P/S, 625 IU/mL IL-7, 85 IU/mL IL-15 (final [cell]= 106 cells/mL) and incubate at 37 &#176;C for 3 days.</li>
	</ol>
	</li>
	<li>Puromycin selection
	<ol>
		<li>Count with AOPI in a fluorescent cell counter.</li>
		<li>Adjust to 1 x 106 cells/mL with T cell growth medium containing IL7 and IL15.</li>
		<li>Add puromycin (2.5 &#181;g/mL) to the media.</li>
		<li>Count cells every 2 days for 6 days and adjust to 1 x 106 cells/mL with cell growth medium containing 3% HS, 1% P/S, 625 IU/mL IL-7, 85 IU/mL IL-15, and 2.5 &#181;g/mL puromycin.</li>
		<li>Check % CAR.</li>
	</ol>
	</li>
	<li>Sort CAR T cell populations and store samples (dry cell pellet) at -80 &#176;C.</li>
</ol>
3. Isolation of gDNA
NOTE: Avoid any cell or gDNA losses throughout the process, as this may compromise the representativity of the sgRNAs. Aim for a sgRNA coverage of at least 500x.
<ol>
	<li>Extract gDNA from samples using the cells and tissue DNA extraction kit. Elute twice to maximize gDNA recovery. 
	NOTE: Do not use more than 4 x 106 cells/column in order to recover the gDNA more efficiently.</li>
	<li>Measure gDNA concentration with the dsDNA quantification kit. 
	NOTE: If the concentration is too low for subsequent steps in the protocol, use solid-phase reversible immobilization (SPRI) bead-based clean-up reagent to concentrate the DNA.</li>
</ol>
4. Enrichment of sgRNA cassette (and elimination of gDNA carryover)
<ol>
	<li>Restriction enzyme digestion
	<ol>
		<li>Digest a maximum of 5 &#181;g gDNA with 20 U of the enzyme/s (e.g., for lentiGuide-puro backbone for Brunello Kinome 1 library use the combination NdeI and PspXI) in a total volume of 50 &#181;L. Scale up the number of reactions to achieve the desired sgRNA representativity. 
		&#8203;NOTE: Select the restriction enzymes to cut in the flanking regions of the cassette, but bear in mind that some of the sgRNAs might contain a restriction site. These sgRNAs will be lost in this step, and they will not show up in the sequencing. Absence of this guide serves as a control of digestion. Supplementary Table 1 shows a list of Kinome 1 sgRNAs that are cleaved by PspXI and NdeI.</li>
		<li>Incubate overnight at 37 &#176;C in a thermocycler.</li>
		<li>Perform a 2x SPRI clean-up.
		<ol>
			<li>Prepare 10 mL of Elution Buffer (EB)&#160;(10 mM Tris-HCl, pH 8.0) and 50 mL of fresh 70% ethanol.</li>
			<li>Vortex the beads and add 100 &#181;L to the product of the digestion.</li>
			<li>Mix thoroughly by pipetting and incubate for 5 min. Magnetize for another 5 min.</li>
			<li>While on the magnet, add 200 &#181;L of 70% ethanol without disturbing the pellet. Wait 30 s, then discard the supernatant.</li>
			<li>Repeat step 4.1.3.3 for a total of 2 washes. Allow the beads to dry for 2 min.</li>
			<li>Remove from the magnet, add 40 &#181;L of elution buffer, and resuspend the beads by pipetting. Incubate for 2 min.</li>
			<li>Magnetize for 2 min and transfer the supernatant to a new tube. 
			NOTE: When setting up this protocol for the first time, verify the digestion through (capillary) electrophoresis. Ensure that a smear is observed.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Pulldown of the sgRNA cassette.
	<ol>
		<li>Preparation of streptavidin magnetic beads.
		<ol>
			<li>Prepare Wash/Binding Buffer 2x: 10 mM Tris-HCl pH 7.5, 2 M NaCl, 1 mM EDTA. Dilute half of the volume with H2O to 1x.</li>
			<li>Vortex the beads for 1 min.</li>
			<li>Transfer the desired volume of beads to a tube (typically 1 mg will suffice), add an equal volume of Wash/Binding Buffer 1x, or at least 1 mL, and resuspend.</li>
			<li>Place the tube on a magnet for 1 min and discard the supernatant.</li>
			<li>Remove the tube from the magnet and resuspend the washed beads in the same volume of 1x Wash/Binding Buffer as the initial volume of beads taken from the vial.</li>
			<li>Repeat for a total of 3 washes.</li>
			<li>Resuspend beads in 2x Wash/Binding Buffer.</li>
		</ol>
		</li>
		<li>Add 5 &#181;L of 10 &#181;M pulldown primers listed in Table 1 to the purified digestion from step 4.1.3.6.</li>
		<li>Incubate in a dry bath at 96 &#176;C for 5 min, then immediately bring to ice for 5 min.</li>
		<li>Add 1 mg of pre-washed streptavidin magnetic beads. 
		NOTE: Biotin-streptavidin binding works best at 1 M NaCl. Make sure to mix&#160;equal volumes of beads and biotinylated DNA.</li>
		<li>Incubate for 20 min while resuspending the mix every 4 min.</li>
		<li>Magnetize for 5 min.</li>
		<li>Wash the beads 3 times with 1&#215; Wash/Binding Buffer.</li>
		<li>Resuspend the beads in 50 &#181;L of EB 8.0. 
		NOTE: The sgRNA cassettes used here are bound to the streptavidin beads. Do not discard them.</li>
	</ol>
	</li>
</ol>
5. sgRNA library preparation
<ol>
	<li>PCR1
	<ol>
		<li>Prepare PCR1 mix with 20 &#181;L of Herculase 5x buffer, 1 &#181;L of dNTP (100 mM), 1 &#181;L of DNA polymerase, 2.5 &#181;L of 10 &#181;M of PCR1 forward and reverse primers listed in Table 1, and 23 &#181;L of PCR grade water. Pipette mix and add PCR1 mix to the beads from step 4.2.8. 
		NOTE: Table 1 lists PCR1 primers designed for the Brunello Kinome Library 1.</li>
		<li>Place the tubes in a thermocycler and run the program for PCR1 described in Table 2. 
		NOTE: Make sure the thermocycler can accommodate 100 &#181;L reactions. Otherwise, aliquot in 50 &#181;L reactions. Scale as needed.</li>
		<li>Pool all PCR1 from the same samples in a single tube.</li>
		<li>SPRI clean-up PCR1 using 150 &#181;L (1.5x) of beads (follow steps 4.1.3.3-4.1.3.7). Resuspend each PCR reaction in 100 &#181;L of EB 8.0. 
		NOTE: If setting up this protocol for the first time, verify the size and quality of the PCR product by running 2 &#181;L of a 30-cycle PCR product in a capillary electrophoresis system (e.g., Agilent&#39;s TapeStation Systems).</li>
	</ol>
	</li>
	<li>PCR2
	<ol>
		<li>Prepare PCR2 mix with 10 &#181;L of the product of PCR1, 10 &#181;L of 5X buffer, 0.5 &#181;L of dNTP (100 mM), 1 &#181;L of DNA polymerase, 1.25 &#181;L of 10 &#181;M PCR2 primers forward and reverse listed in Table 1 (use a different rP7-i7-BC and P5-i5-BC combination for each sample), and 26 &#181;L of PCR grade water for a total volume of 50 &#181;L. 
		NOTE: To maintain library representation, perform one PCR2 reaction per 104 constructs in the library.</li>
		<li>Place the tubes in a thermocycler and run the program for PCR2 described in Table 2.</li>
		<li>SPRI clean-up PCR2&#160;using 75 &#181;L (1.5x) of beads (follow steps 4.1.3.3-4.1.3.7). Resuspend each PCR reaction in 50 &#181;L of EB 8.0.</li>
	</ol>
	</li>
	<li>Quantify PCR2 (i.e., the sgRNA library) using the dsDNA quantification kit and verify the amplicon size by capillary electrophoresis (e.g., TapeStation Systems). Pool the samples.</li>
	<li>Sequence on an Illumina instrument. 
	NOTE: For the 3052 sgRNA Brunello Kinome 1 library here, a sequencing depth of 100-200 million reads per sample is typically enough.</li>
</ol>
6. Analysis
<ol>
	<li>Demultiplex samples using bcl2fastq.</li>
	<li>Check the quality of the fastq files generated with FastQC.</li>
	<li>Extract the sequence of the sgRNA using as a pattern the cassette sequences flanking the sgRNA, as well as the length of the guide (20 nt).</li>
	<li>Align the sequence extracted to the library reference using Bowtie2 to check how many of the hypothetical guides are authentic sgRNA from the library. Use samtools to convert the SAM file with information about the alignment into BAM format and generate summary files.</li>
	<li>Count and normalize the number of reads for each sgRNA in the edited and control samples with the count function from MAGecK software.</li>
	<li>Compare the counts with the test function of MAGecK to obtain the RRA scores and the list of candidate genes.</li>
</ol>

<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>
<li>Redondo-Frutos, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+engineering+in+CAR+T+cells+for+solid+tumors%3A+Current+state%2C+barriers+and+future+developments%5BTitle%5D)+AND+%22Hum+Gene+Ther%22%5BJournal%5D)">Genetic engineering in CAR T cells for solid tumors: Current state, barriers and future developments.</a> Hum Gene Ther. 36 (17-18), 1138-1153 (2025).</li>
<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>
<li>Shang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+CRISPR+screen+identifies+FAM49B+as+a+key+regulator+of+actin+dynamics+and+T+cell+activation%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation.</a> Proc Natl Acad Sci U S A. 115 (17), E4051-E4060 (2018).</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 (2018).</li>
<li>Chen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+CD8%2B+T+cell+CRISPR+screening+reveals+control+by+Fli1+in+infection+and+cancer%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">In vivo CD8+ T cell CRISPR screening reveals control by Fli1 in infection and cancer.</a> Cell. 184 (5), 1262-1280.e22 (2021).</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>Henriksson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+CRISPR+screens+in+T+helper+cells+reveal+pervasive+crosstalk+between+activation+and+differentiation%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Genome-wide CRISPR screens in T helper cells reveal pervasive crosstalk between activation and differentiation.</a> Cell. 176 (4), 882-896.e18 (2019).</li>
<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>
<li>Wei, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+REGNASE-1+programs+long-lived+effector+T+cells+for+cancer+therapy%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy.</a> Nature. 576 (7787), 471-476 (2019).</li>
<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>
<li>Legut, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genome-scale+screen+for+synthetic+drivers+of+T+cell+proliferation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A genome-scale screen for synthetic drivers of T cell proliferation.</a> Nature. 603 (7902), 728-735 (2022).</li>
<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>
<li>Owczarzy, R., Moreira, B. G., You, Y., Behlke, M. A., Wälder, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Predicting+stability+of+DNA+duplexes+in+solutions+containing+magnesium+and+monovalent+cations%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations.</a> Biochemistry. 47 (19), 5336-5353 (2008).</li>
<li>Sasaki, Y., Miyoshi, D., Sugimoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+molecular+crowding+on+DNA+polymerase+activity%5BTitle%5D)+AND+%22Biotechnol+J%22%5BJournal%5D)">Effect of molecular crowding on DNA polymerase activity.</a> Biotechnol J. 1 (4), 440-446 (2006).</li>
<li>Seitz, V., Schaper, S., Dröge, A., Lenze, D., Hummel, M., Hennig, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+method+to+prevent+carryover+contaminations+in+two-step+PCR+NGS+library+preparations%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">A new method to prevent carryover contaminations in two-step PCR NGS library preparations.</a> Nucleic Acids Res. 43 (20), e135(2015).</li>
<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>
<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>
<li>Sui, H., Chen, Q., Imamichi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+pulldown+assay+using+DNA%2FRNA-conjugated+beads+with+a+customized+competition+strategy%3A+An+effective+approach+to+identify+DNA%2FRNA+binding+proteins%5BTitle%5D)+AND+%22MethodsX%22%5BJournal%5D)">A pulldown assay using DNA/RNA-conjugated beads with a customized competition strategy: An effective approach to identify DNA/RNA binding proteins.</a> MethodsX. 7, 100890(2020).</li>
<li>Chaparian, R. R., van Kessel, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Promoter+pulldown+assay%3A+A+biochemical+screen+for+DNA-binding+proteins%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Promoter pulldown assay: A biochemical screen for DNA-binding proteins.</a> Methods Mol Biol. 2346, 165-172 (2021).</li>
</ol>

The authors have no conflicts of interest.

CRISPR screening relies on the principle of single-guide perturbation, meaning that only one sgRNA should be integrated per cell. To achieve this, sgRNAs are typically delivered via lentiviral vectors, and the multiplicity of infection (MOI) must be carefully controlled. An MOI of ca. 0.3 is generally recommended, resulting in 25% to 30% of cells being transduced with a single viral particle6. Accurate titration is therefore essential to determine the volume of viral particles required. Another critical step is the loss of sgRNA coverage during the protocol. Cell loss during harvesting, inefficiencies during gDNA extraction (namely column overloading and incomplete elution of DNA), or excessive template during PCR amplification can all contribute to this loss, hence skewing our sgRNA analysis. Regarding the biotinylated oligos, it is best if they are HPLC purified to avoid that free biotin used in the synthesis saturates the streptavidin beads. Moreover, a 3&#39; amino-modifier is strongly suggested to prevent amplification from the biotinylated primers in subsequent PCRs. Also, when designing these oligos, avoid any overlap with PCR1 primers to prevent competition during amplification. In addition, bioinformatic analysis requires careful attention: sgRNA extraction patterns depend on the plasmid backbone (e.g., lentiGuide-puro or lentiCRISPR v2), and alignment stringency can range from exact matching to some tolerance of mismatches. Furthermore, since this protocol includes a digestion step, some sgRNAs containing restriction sites may be lost, requiring adjustment of the reference library.
In this study, we propose an optimized CRISPR screening library preparation protocol that enriches sgRNA-containing fragments through digestion and, more importantly, a pulldown step. Restriction enzyme digestion has been previously suggested as a strategy to reduce gDNA input and enrich for sgRNA cassettes27. Similarly, biotinylated probes have been widely applied in molecular biology to capture specific nucleic acids or protein-nucleic acid complexes28,29,30,31.
Our protocol builds on these principles and adapts them for CRISPR screening in primary CAR T cells, although several limitations should be acknowledged. First, primary CAR T cells impose constraints on screening scale, as their expansion capacity is limited. To maintain adequate coverage and sgRNA representation, large numbers of starting cells are required. When this is not feasible, smaller custom libraries may be used12, though this reduces the breadth of genetic interrogation. Biological variability is another challenge; differences between donors are expected, and the overlap of significant hits is often modest17. Second, the enrichment strategy relies on the presence and position of restriction enzyme cutting sites. In rare cases, these sites may occur within sgRNA sequences, which is more problematic for small libraries with limited redundancy. However, most libraries, particularly genome-wide designs, include multiple sgRNAs per target, minimizing the overall impact of guide loss. Finally, alternative fragmentation-based enrichment strategies, such as sonication coupled with probe capture24, may provide some flexibility in cases where restriction enzyme digestion is not suitable.
Overall, the sgRNA cassette represents a tiny fraction of the total genomic DNA in CRISPR-edited cells, making efficient enrichment critical for successful library preparation. Compared with standard protocols, our optimized workflow reduces gDNA carryover and favours PCR amplification of the sgRNA of interest. While the combination of gDNA fragmentation and biotinylated probe pulldown with streptavidin beads is not conceptually new, its integration into CRISPR screening avoids common PCR pitfalls and increases CRISPR screen reproducibility. Moreover, incorporating standardized QC metrics at each key step (such as post-sorting T-cell purity, the proportion of puromycin-resistant cells, gDNA integrity, and CAR transduction efficiency) would further enhance the robustness and reproducibility of the workflow. In conclusion, although developed in the context of primary CAR T cells and with only two donors (that may represent a limitation, although several samples from each donor were used), this optimized protocol may be broadly applicable to other CRISPR screenings and NGS workflows with large gDNA carryovers.

Immunotherapy has revolutionized cancer treatment, offering novel strategies to harness and modulate the immune system. Among these, chimeric antigen receptor (CAR) T cell therapy has emerged as one of the most transformative approaches. This therapy involves the genetic engineering of a patient&#39;s T cells ex vivo with a synthetic receptor designed to recognize a specific tumor antigen, thereby enhancing antitumor activity1. CAR T cells have shown remarkable success in hematological malignancies, with outcomes ranging from short-lived remissions to prolonged disease-free survival with minimal toxicity in patients receiving CD19 or BCMA-targeted therapies2. To date, the FDA has approved seven CAR T cell products for hematological cancers3. Despite these advances, significant limitations remain. Challenges arise from the CAR T cell product itself, often influenced by the manufacturing process or the fitness of the patient&#39;s T cells, which may be impaired by disease progression, prior therapies, or age. Tumor-intrinsic resistance mechanisms, such as antigen downregulation, can also compromise efficacy4. Furthermore, the immunosuppressive tumor microenvironment poses an additional barrier that severely limits CAR T cell persistence and function, particularly in patients with solid tumor5. Consequently, there is an urgent need to enhance both the initial and long-term efficacy of CAR T cell therapies.
The advent of CRISPR-Cas9 gene editing has provided powerful tools to dissect gene function through large-scale screening approaches. In these assays, cells are transduced with a single-guide RNA (sgRNA) library, typically delivered by lentiviral vectors, ensuring one sgRNA per cell and stable genomic integration. Following Cas9-mediated editing and selection of transduced cells, genomic DNA (gDNA) is extracted, and sgRNA cassettes are amplified by PCR for library preparation. High-throughput sequencing then enables the quantification of sgRNA distributions across different phenotypes, thereby identifying genes that positively or negatively regulate the process under study6.
CRISPR screening has been widely applied to explore T cell biology and, more recently, to enhance CAR T cell performance. Genome-wide screens have identified regulators of fundamental T cell processes, including activation, proliferation, and differentiation. For example, FAM49B was identified as a regulator of T cell receptor signaling7, while SOCS1, TCEB2, RASA2, and CBLB were shown to be essential for proliferation following stimulation8. Beyond these core pathways, CRISPR screening has also elucidated genes involved in T cell memory and exhaustion. In vivo studies identified Fli1 as a candidate to enhance effector responses without disrupting memory or exhaustion precursors9, and the chromatin remodeler Arid1a as a regulator whose loss reduces T cell exhaustion10. Regulators of T helper type 2 (Th2) differentiation have also been characterized with this approach11. Using a custom sgRNA library targeting 25 kinases, p38 was found to promote expansion, memory formation, and protection from oxidative and genomic stress12. Similarly, REGNASE-1 knockout in CD8+ T cells conferred a long-lived effector phenotype that improved tumor control in melanoma and leukemia models13. Additional genome-wide screens identified Dhx37 as a regulator of T cell activation and cytotoxicity14, while LTBR and other genes were validated as enhancers of T cell function in CAR T and &#947;&#948; T cells15. More recently, CRISPR screening has been applied directly in CAR T cells, revealing novel targets such as PRODH2, an enzyme involved in proline metabolism that enhances CAR T antitumor activity16, and TLE4 and IKZF2, whose inactivation improved CAR T efficacy in glioblastoma models17.
A critical step in CRISPR screens is precisely the selective amplification of sgRNA cassettes from exceedingly large amounts of gDNA, a challenge difficult to circumvent given the vast number of cells used in CRISPR screenings. PCR amplification from such large amounts of DNA carryover poses several technical challenges, including molecular crowding that limits the physical diffusion of DNA and polymerase molecules, transient non-specific binding of the primers and the polymerase to the DNA background, off-target amplification resulting in primer depletion, or Mg2+ sequestration by the DNA phosphate backbone, among other challenges18,19,20,21. These often result in PCR failure and/or bias. To mitigate these issues, different strategies have been proposed, including separating sgRNA amplification from adaptor addition into two PCR steps22, using target enrichment with biotinylated oligos and magnetic bead capture23,24, or designing plasmids with restriction sites flanking the sgRNA cassette for fragment enrichment25.
In this study, we present an optimized CRISPR-Cas9 knockout screening protocol for primary human CAR T cells. The CRISPR library used is Brunello Kinome 1, a library that contains 3052 unique 20nt-long sgRNAs targeting 763 human kinase genes (4 sgRNAs per target). T cells were isolated through CD4+ and CD8+ magnetic positive selection from the peripheral blood mononuclear cells (PBMC)-enriched fraction of blood samples from healthy donors. Upon activation with anti-CD3 and anti-CD28, T cells were spinfected with the CRISPR screening library, then transduced with the CAR lentivirus. Cells were expanded, then nucleofected to introduce the Cas9 protein. CRISPR-bearing cells were selected with puromycin, and CAR-positive T cells were sorted. Following extraction, genomic DNA was digested with restriction enzymes (RE), and the sgRNA-containing fragments were pulled down by biotin probe-streptavidin capture. Next, sgRNA were selectively amplified and indexed via two consecutive PCRs, and the resulting libraries were sequenced. By incorporating an intermediate step to reduce gDNA carryover, we were able to selectively retrieve and amplify our sgRNA of interest, which allows us to study the role of the kinases in CAR T cells.

<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&amp;nbsp;</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>autoMACS NEO Separator</td><td>Miltenyi</td><td>130-120-327</td><td>High-speed magnetic cell sorter</td></tr><tr><td>Biotinylated oligos</td><td>IDT</td><td></td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A9647</td><td></td></tr><tr><td>Cellometer K2 Fluorescent Cell Counter</td><td>Revvity</td><td>CMT-K2-MX-150</td><td>Fluorescent cell counter</td></tr><tr><td>CHT4 Counting Chambers</td><td>Revvity</td><td>CHT4-SD100-002</td><td></td></tr><tr><td>CytoSinct CD4 Nanobeads</td><td>GenScript</td><td>L00863-1</td><td></td></tr><tr><td>CytoSinct CD8 Nanobeads</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>DynaMag-2 magnet</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>Ethanol</td><td>Merck</td><td>1,00,98,31,000</td><td></td></tr><tr><td>ExPERT Atx Electroporator</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 &amp;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>Herculase Buffer 5X</td><td>Agilent</td><td>600675-52</td><td>5X buffer</td></tr><tr><td>Herculase II Fusion Enzyme</td><td>Agilent</td><td>600679-51</td><td>DNA polymerase</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>MaxCyte Electroporation Buffer</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>Nucleospin Tissue</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>Propidium Iodine</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>Puromycin</td><td>Gibco</td><td>A11138-03</td><td></td></tr><tr><td>Qubit 3.0 Fluorometer</td><td>Invitrogen</td><td>Q33216</td><td></td></tr><tr><td>Qubit HS DNA assay</td><td>Invitrogen</td><td>Q32851</td><td></td></tr><tr><td>R-50x3 Sterile Well Processing Assembly</td><td>MaxCyte</td><td>LM243882</td><td></td></tr><tr><td>T Cell TransAct human</td><td>Miltenyi</td><td>130-111-160</td><td>T cell stimulation reagent</td></tr><tr><td>Tape Station 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>T cell growth medium</td></tr><tr><td>Tris Buffer, 1.0 M, pH 8.0, Molecular Biology Grade</td><td>EMD Milipore</td><td>648314</td><td></td></tr><tr><td>Water, for molecular biology, DNAse, RNAse and Protease free</td><td>ThermoScientific</td><td>327390010</td><td></td></tr></tbody></table>

improving crispr-cas9 screens in car t cells: a refined method for library preparation

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

Chimeric antigen receptor (CAR) T cell therapies have demonstrated remarkable efficacy in several hematological malignancies, yet their success has not been fully replicated in solid tumors. Moreover, even in hematological cancers, relapse after CAR T cell infusion continues to compromise long-term outcomes. These challenges highlight the urgent need to develop strategies that enhance CAR T cell efficacy, persistence, overcoming tumor and microenvironment-mediated resistance. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based screening platforms provide a powerful approach to systematically identify genes that regulate CAR T cell function. By linking genetic perturbations to phenotypic outcomes, these assays enable the discovery of pathways controlling activation, proliferation, memory formation, and cytotoxicity. Standard workflows involve transduction of substantial numbers of cells with a single guide RNA (sgRNA) library, Cas9-mediated editing, selection of edited cells, and PCR amplification of sgRNA cassettes from genomic DNA (gDNA) prior to sequencing. However, PCR amplification using large amounts of gDNA poses significant challenges and often fails to selectively amplify and retrieve sgRNAs. Here, we describe an optimized CRISPR-Cas9 knockout screening protocol, which we have tested on primary human CAR T cells. The method here incorporates an intermediate step during sgRNA library preparation that reduces gDNA carryover through enzymatic digestion and selective pulldown of the sgRNA cassette, thereby increasing the efficiency of the first PCR amplification. This modification allowed us to retrieve sgRNA information across our CAR T cell screens, which had remained elusive in our previous attempts using traditional 1 and 2-step PCR amplification protocols. In conclusion, this optimized workflow facilitates CRISPR screening library preparation in challenging samples and enables the identification of key genetic determinants that can be targeted to improve therapeutic efficacy.

As described in the Protocol Section, CAR T cells were generated following our established protocol26. For CRISPR screening, we adapted the workflow developed by Wang et al.17 (Figure 1A). Briefly, T cells from two independent donors were isolated and activated for 24 hours before consecutive transduction with both the CRISPR sgRNA library and the CAR lentiviral vector, reaching 67 and 72% CAR-positive cells at day 5. A fraction of cells was nucleofected with Cas9 protein to induce knockouts, while the remaining cells served as a baseline for sgRNA representation. Transduced cells were selected with antibiotics, and distinct populations were subsequently sorted according to the phenotypes under study. CAR T cell proliferation was monitored throughout the screen using AOPI-based cell counts and expressed as fold change (Figure 1B). Following puromycin supplementation at days 8, 10, and 12, we confirmed successful enrichment of CAR T cells transduced with the CRISPR library in both donors, as evidenced by continued survival and proliferation. Untransduced T cells (UTD) and CAR T cells lacking CRISPR library transduction were used as controls of antibiotic selection.
After genomic DNA extraction, sgRNA libraries were prepared via PCR amplification. To overcome issues arising from excessive gDNA carryover, we implemented a protocol incorporating restriction enzyme digestion and biotin-streptavidin pulldown of the sgRNA cassette (Figure 2A,B). gDNA was digested with restriction enzymes NdeI and PspXI targeting sites flanking the sgRNA cassette (Figure 2C). Biotinylated oligos, designed to bind upstream of PCR1 primer annealing sites, were used to capture sgRNA cassette fragments, then pulled down with streptavidin beads to eliminate carryover gDNA. Fragments containing sgRNA were then selectively amplified (Figure 2D) and indexed through 2 consecutive PCRs.
Libraries from each sample were sequenced using Next Generation Sequencing (NGS). After demultiplexing, quality control, and sgRNA identification, data were analyzed with the MAGeCK pipeline. First, the count command quantified and normalized sgRNA read counts in control (basal) and Cas9-edited samples (Figure 3A,B). Pearson and Spearman correlations indicate that the representativity of sgRNA was maintained during the experiment (donor gDNA) compared with the plasmidic library (Figure 3C). Next, the test command compared both conditions, assigning robust rank aggregation (RRA) scores to each gene (Figure 3D). Significantly enriched or depleted genes were identified using thresholds of p &lt; 0.05 and log fold change (LFC) &gt; 0.5 (Figure 3E). Finally, pathway enrichment analysis of candidate genes was performed using Metascape, revealing top Gene Ontology terms associated with the identified regulators (Figure 3F).
<img alt="CRISPR gene editing flowchart, T cell modification timeline, phenotype variation graph, fold change chart." class="xfigimg" src="/files/ftp_upload/69721/69721fig1.jpg" data-altupdatedat="1780372819000" data-alt="Figure 1"> 
Figure 1: CRISPR screening in CAR T cells. (A) Scheme of the CRISPR screening in CAR T cells. (B) Selection of transduced cells with puromycin (P). Plasmidic libraries include a puromycin resistance gene for positive selection. Antibiotic was added at day 8 to the media in non-transduced samples of untransduced (UTD) and CAR T cells and transduced CAR T cells. Samples without puromycin were used as a control of proliferation. Data from two independent donors is shown. <a href="https://www.jove.com/files/ftp_upload/69721/69721fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Gene editing process; A: digestion→pulldown→PCR, B: gRNA schematic, C/D: electrophoresis results." class="xfigimg" src="/files/ftp_upload/69721/69721fig2.jpg" data-altupdatedat="1780372819000" data-alt="Figure 2"> 
Figure 2: Library preparation for sgRNA enrichment analysis. (A) Scheme of the protocol for library preparation. (B) Scheme of the relative localization of the cut sizes of the restriction enzyme (scissors) and the annealing regions of PCR1 primers (arrow) and biotinylated primers (arrow with green dot). (C) Electrophoresis of the gDNA pre- and post-RE digestion. (D) Selective enrichment of sgRNA cassettes after PCR1. Data from two independent donors from basal and Cas9 samples of the two phenotypes is shown. <a href="https://www.jove.com/files/ftp_upload/69721/69721fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Comparative gene expression analysis: tables, charts, correlation plots, log fold change, significance analysis." class="xfigimg" src="/files/ftp_upload/69721/69721fig3.jpg" data-altupdatedat="1780372819000" data-alt="Figure 3"> 
Figure 3: Guide enrichment analysis with MAGeCK. (A) Summary of the output of the MAGeCK count function. (B) Distribution of read counts. (C) Pearson and Spearman coefficients for correlation between donor gDNA and plasmid library counts. (D) Potential enriched/depleted genes ranked by a modified robust ranking aggregation (RRA score). (E) Volcano plot showing significant genes (p-value &lt; 0.05, LFC &gt; 0.5) in red. (F) Top Gene Ontology (GO) terms after Metascape pathway analysis of the enriched significant genes. <a href="https://www.jove.com/files/ftp_upload/69721/69721fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Sequence of primers. <a href="https://www.jove.com/files/ftp_upload/69721/Table%201.xls" target="_blank">Please click here to download this Table.</a>
Table 2: PCR conditions. <a href="https://www.jove.com/files/ftp_upload/69721/TABLE%202.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Table 1: List of sgRNA in Kinome 1 library and sgRNAs lost during digestion with NdeI and PspXI <a href="https://www.jove.com/files/ftp_upload/69721/Supplementary%20Table%201.xlsx" target="_blank">Please click here to download this File.</a>

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.

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

Our research focuses on using CRISPR screening approaches to identify targets and enhance the effectiveness of CAR T cell therapies. Current experiment challenges includes achieving reproducible laboratory methods in CRISPR screening approaches. To begin, add a predetermined amount of Lenta viral CRISPR library to a 24 well plate containing T cells.Add poly brain at a final concentration of eight micrograms per milliliter. Mix gently to ensure even distribution. Centrifuge the cells at 700 G for 90 minutes at 32 degrees Celsius to perform spin-fection.After centrifugation, carefully remove the plate from the centrifuge. Replace the media with fresh T-cell growth medium. After six hours, add the CAR lenta viral vector at a multiplicity of infection of three.Incubate the cells at 37 degrees Celsius for four days. Once incubation is complete, collect the CAR T cells. Wash once with PBS.Then stain the cells using acradine orange propidium iodide, and count them in a fluorescent cell counter. Prepare a four micromolar solution of cas9 in electroporation buffer. Resuspend five times 10 to the power six CAR T cells in 50 microliters of cas9 solution.Then pipette 50 microliters of the cell suspension into each well of a multi-well cuvete strip without forming bubbles. Remove any visible bubbles by gently running a pipette tip along the edges of the well. Use the expand T-cell three program to nucleoffect the CAR T cells.Place the cuvettes in an incubator at 37 degrees Celsius for 40 minutes. Resuspend the nucleoffected cells in T-cell growth medium. Then incubate again at 37 degrees Celsius for three days.Count the cells using acradine orange propidium iodide staining in a fluorescent cell counter. After adjusting the cell density to ten to the power of six cells per milliliter, add puromycin to a final concentration of 2.5 micrograms per milliliter. After six days of incubation, assess the percentage of CAR positive cells using flow cytometry.Then sort CAR T cell populations and store dry cell pellets at minus 80 degrees Celsius. Extract genomic DNA from the samples using a cell and tissue DNA extraction method. Measure genomic DNA concentration using a double stranded DNA quantification kit.Next digest up to five micrograms of genomic DNA with 20 units of the selected restriction enzymes in a total volume of 50 microliters. Incubate overnight at 37 degrees Celsius in a thermocycler. The next day, prepare 10 milliliters of elution buffer containing 10 millimolartris HCL and 50 milliliters of fresh 70%ethanol.Pipette 100 microliters of the digested product into a tube containing vortexed beads. Pipette to mix the suspension thoroughly and incubate for five minutes at room temperature before magnetizing for another five minutes. While on the magnet, discard the supernatant and pipette 200 microliters of the 70%ethanol solution without disturbing the pellet.After 30 seconds, discard the supernatant and perform two more alcohol washes. After two washes, allow the beads to dry for two minutes, then remove them from the magnet. Add 40 microliters of elucian buffer and pipette to resuspend the beads.Magnetize the suspension for two minutes, then transfer the supernatant into a new tube. Add one x wash binding buffer to a tube containing one milligram of vortex streptavidin magnetic beads. Pipette to resuspend the beads in the buffer.Then place the suspension on a magnet for one minute, discard the supernatant and perform two more washes. After the third wash reuspend the beads in two x wash binding buffer. Then add five microliters of 10 micromolar biotinylated pull down primers to the purified digestion product.Incubate the mixture in a dry bath at 96 degrees Celsius for five minutes and immediately cool on ice for five minutes. Add one milligram of pre-washed, streptavidin magnetic beads to the reaction, ensuring that both the reaction and the streptavidin beads have equal volumes. Incubate for 20 minutes at room temperature while resuspending every four minutes.Magnetize the beads for five minutes, then wash three times with one x wash binding buffer. Finally, resuspend the beads containing bound sgRNA cassette in 50 microliters of elucian buffer. Prepare and amplify the sgRNA library and sequence it.Following antibiotic selection, CAR T cells transduced with the CRISPR library showed continued survival and proliferation in both donors after puromycin treatment. Untransduced T-cell, and CAR T cells lacking CRISPR library transduction did not show comparable proliferation under antibiotic selection and served as negative controls. Restriction enzyme digestion with NDEL and PSPXI generated fragmented genomic DNA while preserving the single guide RNA cassette for enrichment.Fragments containing sgRNA were then selectively amplified. Pearson and Spearman correlations showed that single guide RNA read counts and library complexity were maintained in donor genomic DNA samples compared with the plasmid library. Robust rank aggregation analysis identified genes that were significantly enriched or depleted following Cas9 editing in both donors.Pathway enrichment analysis revealed top gene ontology terms associated with the identified genetic regulators. Our protocol offers a reliable alternative to existing laboratory techniques using CRISPR screening approaches. Our findings will advance the field by supporting the development of more reproducible and robust CRISPR screening protocols.Our results paved the way for identifying genes and can be moderated to enhance CAR T cell phenotype and function.

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

Research

JoVE Journal

Immunology and Infection

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

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Here, we present a protocol to optimize CRISPR-Cas9 to achieve a higher specificity without the loss of on-target activity. We use a directed evolution approach called Sniper-screen to find a mutant Cas9 with the desired characteristics. Sniper-Cas9 is compatible with truncated single-guide RNAs and delivery in a ribonucleoprotein format, well-known strategies for achieving higher specificities.

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Here we describe a rapid and direct in vivo CRISPR/Cas9 screening methodology using ultrasound-guided in utero embryonic lentiviral injections to simultaneously assess functions of several genes in the skin and oral cavity of immunocompetent mice.

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

Production of Human CRISPR-Engineered CAR-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.

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

Discover an optimized CRISPR-Cas9 screening protocol for primary human CAR T cells using enzymatic digestion and selective pulldown. This method improves sgRNA recovery from genomic DNA to identify genetic regulators enhancing CAR T cell therapies.

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

Explore More Videos