REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
c-Myc Monoclonal Antibody (9E10) Dilution Factor: 1:100	Invitrogen	Cat# MA1-980; RRID: AB_558470
Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) Dilution Factor: 1:1000	Cell Signaling Technology	Cat# 4410
APC anti-human CD3 antibody Dilution Factor: 1:20	BioLegend	Cat# 300412; RRID: AB_314066
FITC anti-human beta2-microglobulin antibody Dilution Factor: 1:20	BioLegend	Cat# 316304; RRID: AB_492837
PE anti-human CD279 (PD-1) antibody Dilution Factor: 1:20	BioLegend	Cat# 329906; RRID: AB_940483
PE/Cyanine7 anti-human CD152 (CTLA-4) antibody Dilution Factor: 1:20	BioLegend	Cat# 369613; RRID: AB_2632875
Bacterial and virus strains
NEB® 5-alpha Competent E. coli (High Efficiency)	New England Biolabs	Cat# C2987I
BL21(DE3) Competent E. coli	New England Biolabs	Cat# C2527H
Biological samples
Human PBMC, Female donor ranging from 25-35 years old	N/A	N/A
Chemicals, peptides, and recombinant proteins
Isopropyl β-D-1-thiogalactopyranoside	Inalco Pharmaceuticals	Cat# 1758-1400
Tryptone	Oxoid	Cat# LP0042B
Yeast extract	Oxoid	Cat# LP0021B
Spectinomycin	Inalco Pharmaceuticals	Cat# 1758-9311
Ampicillin	Inalco Pharmaceuticals	Cat# 1758-9314
NaCl	Biodee	Cat# DE0008
Ni-NTA Agarose	QIAGEN	Cat# 30230
Tris	Biodee	Cat# DE0006
KCl	Sigma-Aldrich	Cat# P5405
Imidazole	Sigma-Aldrich	Cat# I5513
Phenylmethylsulfonyl fluoride	Sigma-Aldrich	Cat# 78830
Electroporation buffer	Celetrix LLC, Manassas VA	Cat# 13-0104
0.4% Trypan Blue Solution	Gibco	Cat# 15250061
Ficoll-Paque PLUS	Cytiva	Cat# 17-1440-02
XYbeads Human CD3/CD28 T Cell Activator	SAILY BIO	Cat# XY-B002-001
X-VIVOTM15	Lonza	Cat# 04-418Q
Fetal Bovine Serum	Gibco	Cat# 10099
Fetal Bovine Serum	PAN-Biotech	Cat# P30-3302
Dulbecco’s Modified Eagle’s Medium	MACGENE	Cat# 15019
Trypsin-EDTA (0.25%) phenol red	MACGENE	Cat# CC017
Penicillin-Streptomycin solution	Hyclone	Cat# SV30010
KOD-One PCR Master Mix	TOYOBO	KMM-101
PBS	MACGENE	Cat# CC008
Opti-MEM™ I Reduced Serum Medium	Thermo Fisher Scientific	Cat# 31985070
Benzonase Nuclease	Smart Lifesciences	Cat# SLP00800
DNase I, RNase-free	Thermo Fisher Scientific	Cat# EN0521
Bestar® Sybr Green qPCR Master Mix	DBI® Bioscience	Cat# DBI-2043
RPMI-1640	MACGENE	Cat# CM10040
IL-2 Protein, Human, Recombinant	SinoBiological	Cat# 11848-HNAE
IL-7 Protein, Human, Recombinant	SinoBiological	Cat# 11821-HNAE
IL-15 Protein, Human, Recombinant	SinoBiological	Cat# 10360-HNCE
BSA	YEASEN	Cat# 36103ES25
EDTA	Sigma-Aldrich	Cat# E9884
PEI MAX® - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000)	Polysciences	Cat# 24765
Critical commercial assays
Dynabeads™ Untouched™ Human T Cells Kit	Invitrogen	Cat# 11344D
Human IFN gamma Uncoated ELISA kit	Invitrogen	Cat# 88-7316-88
Human TNF alpha Uncoated ELISA kit	Invitrogen	Cat# 88-7346-88
LDH-Glo™ Cytotoxicity Assay	Promega	Cat# J2380
DNA Clean & Concentrator-25 (Capped)	Zymo Research	Cat# D4033
FastPure Cell/Tissue DNA Isolation Mini Kit	Vazyme	Cat# DC102
Experimental models: Cell lines
NALM-6 Passage numbers: 1–20	ATCC	CRL-3273™
AAV293 cells Passage numbers: 1–20	Agilent	Cat# 240073
Oligonucleotides
ITR-FW	Genewiz	5′- GGAACCCCTAGTGATGGAGTT
ITR-RV	Genewiz	5′-CGGCCTCAGTGAGCGA
TRAC-1st-FW	Genewiz	5′- CCCTTGTCCATCACTGGCAT
CART-PCR-FW	Genewiz	5′- GGAGTACGACGTGCTGGATAAG
TRAC-2st-RV	Genewiz	5′- GCACACCCCTCATCTGACTT
DBCO modified crRNA target TRAC	GENERAL BIOL	5′-AAUAAUUUCUACUCUUGUAGAU GAGUCUCUCAGCUGGUACAC
Recombinant DNA
pUltra-CNF (Data S1)	Gifted by Prof. Peter G. Schultz	Addgene 48215
pET22B-AsCas12a-2C-NLS (Data S1)	This paper	N/A
pAAV CD19-CAR (Data S1)	This paper	N/A
pHelper (Data S1)	Agilent	Cat# 240071
pAAV-RC6 (Data S1)	Cell Biolabs, Inc.	Cat# VPK-426
Software and algorithms
Tanon Gis	Tanon	N/A
CytExpert 2.3	Beckman Coulter	https://cytexpert.software.informer.com/2.3/
Gen5	BioTek	https://www.biotek.com/products/software-robotics-software/gen5-microplate-reader-and-imager-software/
QuantStudio™ Real-Time PCR Software	Thermo Fisher Scientific	https://www.thermofisher.com/jp/ja/home/global/forms/life-science/quantstudio-6-7-flex-software.html
ÄKTA pure protein purification system	Cytiva	https://www.cytivalifesciences.com/en/us/shop/chromatography/chromatography-systems/akta-pure-p-05844
GraphPad 6.01	GraphPad	https://www.graphpad.com/dl/96314/10B92408/
Deposited data
Plasmid map	This paper	Mendeley Data: https://doi.org/10.17632/x7jmxr2497.1)
Other
Amicon® Ultra-15 Centrifugal Filter Units 50K	Millipore	Cat# UFC905024
AzF (Unnatural amino acid)	Toronto Research Chemicals	A922600
NAEK (Unnatural amino acid)	SIRIUS FINE CHEMICALS	SC-8027
AeF (Unnatural amino acid)	Chemical synthesized	N/A
Millipore membrane filter, 0.22 mm pore size	Merck	Cat# SLGVR04NL
Superdex® 200 increase 10/300 GL	Cytiva	Cat# 28990944
Electroporator	Celetrix LLC, Manassas VA	CTX-1500A LE
Electroporation tube	Celetrix LLC, Manassas VA	Cat# 12-0107
DynaMag™-5 Magnet	Invitrogen	Cat# 12303D
CytoFLEX LX Flow Cytometer	Beckman Coulter	N/A
Synergy H1 Hybrid Multi-Mode Reader	BioTek	Synergy™ H1
NanoDrop One Microvolume UV-Vis Spectrophotometer	Thermo Fisher Scientific	ND-ONE-W