Jun 01, 2026
Base Editing Protocol: Disruption of PDCD1, TIGIT, and TRAC Loci in Primary T Cells Using Adenine and Cytosine Base Editors Delivered as IVT mRNA
- Jethro Langley1,
- Jada Curry1,
- Lou Baudrier1,
- Pierre Billon1
- 1University of Calgary
Protocol Citation: Jethro Langley, Jada Curry, Lou Baudrier, Pierre Billon 2026. Base Editing Protocol: Disruption of PDCD1, TIGIT, and TRAC Loci in Primary T Cells Using Adenine and Cytosine Base Editors Delivered as IVT mRNA. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzwmx8vx1/v1
Manuscript citation:
Zemp et al. 2026. GPNMB-directed CAR T-cell therapy against MiT/TFE family fusion-driven solid tumors. Nature Cancer. Accepted.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 28, 2026
Last Modified: June 01, 2026
Protocol Integer ID: 318098
Keywords: adenine base editing, cytosine base editing, cytosine base editor, ivt mrna targeted gene knockout of pdcd1, ivt mrna, base editing protocol, lentiviral car transduction, following lentiviral car transduction, targeted gene knockout, using adenine, base editor, mrna, edited car, modified synthetic grna, synthetic grnas by neon electroporation
Abstract
Targeted gene knockout of PDCD1 (PD-1), TIGIT, and TRAC in activated primary human T cells via adenine base editing (ABE) or cytosine base editing (CBE) to disrupt splice donor/acceptor sites. Base editors are delivered as in vitro-transcribed (IVT) mRNA together with chemically modified synthetic gRNAs by Neon electroporation. This protocol generates mock-edited or multiplex base-edited CAR-T cells following lentiviral CAR transduction.
Guidelines
- Use ABE mRNA with PDCD1 or TIGIT gRNAs and CBE mRNA with the TRAC gRNA. Multiplex editing (e.g., simultaneous PDCD1 + TIGIT) can be performed by combining the appropriate mRNAs and gRNAs in a single electroporation reaction; total mRNA and gRNA amounts should be optimized.
- The AAVS1 gRNA serves as a non-targeting mock control.
- All IVT steps must be performed under RNase-free conditions.
- Electroporation parameters (1600 V, 10 ms, 3 pulses) are optimized for 1 × 10^6 T cells in 100 μL; viability and editing efficiency should be verified for each donor.
- Flow cytometry and Sanger sequencing are complementary methods; Sanger/EditR provides nucleotide-level editing efficiency at the target splice site, while flow cytometry provides protein-level verification of disruption.
- This protocol is performed on day 3 post-activation following lentiviral CAR transduction; refer to the “CAR Generation and Preclinical LV Vector Manufacturing” protocol for upstream steps.
- Include unedited, mock-electroporated, and single-edited controls in every experiment.
- Base editor mRNA can be stored at −80°C for long-term use; avoid repeated freeze-thaw cycles.
Materials
- gRNAs: chemically modified Alt-R CRISPR-Cas9 sgRNAs (IDT)
- PDCD1 (ABE, splice-site disruption): 5’-CACCUACCUAAGAACCAUCC-3’
- TIGIT (ABE, splice-site disruption): 5’-CAGGCCUUACCUGAGGCGAG-3’
- TRAC (CBE, splice-site disruption): 5’-UUCGUAUCUGUAAAACCAAG-3’
- AAVS1 (mock control): 5’-AGAGCUAGCACAGACUAGAG-3’
- Cytosine base editor (CBE) IVT template: Addgene #193843
- Adenine base editor (ABE) IVT template: Addgene #193844 with CBE coding sequence replaced with ABE8e from pCMV-MMLVgag-3xNES-ABE8e (Addgene #181751)
- IVT template PCR primers:
- Forward: 5’-TCGAGCTCGGTACCTAACTACGACTCACTATAAGG-3’
- Reverse (poly(T)-tailed): 5’-T(113)CTCCTACTCAGGCTTTATTCAAAGACCA-3’
- Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, cat. no. M0493)
- PCR Cleanup Kit (Geneaid, cat. no. DFH300)
- Nuclease-free water (Invitrogen, cat. no. 10977015)
- HiScribe® T7 High Yield RNA Synthesis Kit (New England Biolabs, cat. no. E2040)
- N1-methyl-pseudouridine-5’-triphosphate (TriLink BioTechnologies, cat. no. N-1081-1)
- CleanCap® Reagent AG (TriLink BioTechnologies, cat. no. N-7113-1)
- Monarch® Spin RNA Cleanup Kit (New England Biolabs, cat. no. T2040L)
- Protector RNase Inhibitor (Millipore Sigma, cat. no. 3335399001)
- Neon Buffer R (Thermo Fisher Scientific)
- ImmunoCult-XF medium (StemCell Technologies)
- Recombinant human IL-7 and IL-15 (10 ng/mL each)
- Flow cytometry antibodies: anti-PD-1 (BioLegend, cat. no. 329905), anti-TIGIT (BioLegend, cat. no. 372713), anti-TCRα/β (BioLegend, cat. no. 306722)
- Locus-specific Sanger PCR primers:
- PDCD1: Forward 5’-TGCTCCAGGCATGCAGATCC-3’, Reverse 5’-CCTGCCAGGGACTGAGAGTG-3'
- TIGIT: Forward 5’-GTGTCTCCTCCTGATCTGGG-3’, Reverse 5’-ACCCCACCTCATTCTCAGAG-3’
- TRAC: Forward 5’-GAGCCAAGTCTAGTCGGTGT-3’, Reverse 5’-GTAGGAGAGTTTGGTGGGCT-3’
Procedure
gRNA Design. Design gRNAs targeting splice donor or splice acceptor sites using SpliceR to enable efficient disruption via adenine or cytosine base editing. Use the gRNA sequences listed above (or the AAVS1 mock control sequence).
Base Editor IVT mRNA Production. Obtain or generate linear IVT templates:
CBE template from Addgene #193843.
ABE template by replacing the CBE coding sequence in the plasmid backbone with ABE8e from Addgene #181751. Amplify and poly(T)-tail the linear templates simultaneously by PCR using the forward and reverse primers listed above. Perform PCR with Q5® Hot Start High-Fidelity DNA Polymerase for 25 cycles. Verify PCR products by agarose gel electrophoresis. Purify products using the PCR Cleanup Kit and elute in nuclease-free water. Perform in vitro transcription using the HiScribe® T7 High Yield RNA Synthesis Kit according to manufacturer’s instructions, except for substituting UTP with N1-methyl-pseudouridine-5’-triphosphate and incorporating a 5’ cap co-transcriptionally with CleanCap® Reagent AG. Purify IVT mRNA using the Monarch® Spin RNA Cleanup Kit and resuspend in 25 μL nuclease-free water. Adjust mRNA concentration to 2 μg/μL, aliquot, and store at −80°C.
T-Cell Preparation. Isolate, activate, and transduce primary T cells with lentiviral vectors encoding CAR constructs as described in the “CAR Generation and Preclinical LV Vector Manufacturing” protocol. On day 3 post-activation, wash cells twice with PBS and resuspend at 1 × 10^6 cells per 100 μL in Neon Buffer R.
Electroporation. For each reaction, combine 1 × 10^6 T cells (in 100 μL Neon Buffer R) with 4.5 μg base editor mRNA (ABE mRNA for PDCD1/TIGIT gRNAs; CBE mRNA for TRAC gRNA), 100 pmol of the corresponding gRNA, and 2.5 μL of Protector RNase Inhibitor. Electroporate using the Neon Transfection System (100 μL tip) under the following conditions:
Voltage: 1600 V
Pulse width: 10 ms
Number of pulses: 3 Immediately after electroporation, transfer cells to pre-warmed ImmunoCult-XF medium supplemented with 10 ng/mL IL-7 and IL-15.
Post-Electroporation Culture. Culture edited cells in ImmunoCult-XF medium + IL-7/IL-15 at 37°C, 5% CO2 until analysis.
Base Editing Efficiency Assessment. (≥72 hours post-electroporation)
Flow Cytometry. Stain cells with anti-PD-1, anti-TIGIT, or anti-TCRα/β antibodies to quantify surface protein knockout efficiency.
Sanger Sequencing Validation. Extract genomic DNA from edited cells. Amplify target loci by PCR using the locus-specific primer pairs listed above. Verify PCR products by agarose gel electrophoresis. Purify products using the PCR Cleanup Kit and submit for Sanger sequencing (Azenta). Quantify editing efficiency by decomposition of Sanger chromatograms using EditR.