ArticlePDF Available

CRISPR-Edited Immune Effectors: The End of the Beginning

CRISPR-Edited Immune
Effectors: The End of the
Feiyan Mo,
Helen E. Heslop,
and Maksim Mamonkin
Genome editing has emerged as a powerful
tool to enhance the potency of effector cell
therapies and potentially improve their
Stadtmauer et al.
at the University
of Pennsylvania recently reported prelimi-
nary results of a rst-in-man clinical study
using CRISPR/Cas9-edited therapeutic
T cells in patients with advanced malig-
nancies. In this study, peripheral T cells
were obtained from patients with multiple
myeloma or sarcoma. To allow their engi-
neered T cells to be more resistant to tu-
mor-mediated immune suppression, the
cells were then activated and treated with
a mixture of CRISPR/Cas9 ribonucleopro-
teins (RNPs) to disrupt the PDCD1 gene
that encodes inhibitory receptor PD-1 as
well as TRAC and TRBC genes. TRAC and
TRBC encode constant regions of endoge-
nous TCR aand bchains, respectively. By
eliminating endogenous T cell receptor
(TCR) expression, the authors aimed to in-
crease the expression of the transgenic NY-
ESO-1/LAGE-1-specic TCR they subse-
quently incorporated by lentiviral transduc-
tion. The resulting NY-ESO-1 TCR-trans-
genic CRISPR triple-edited cells, or NYCE
cells,were manufactured for four patients
enrolled in the study, of whom three were
infused. Even though the number of patients
treated with NYCE T cells remains insuf-
cient to draw denitive conclusions about
the safety of their approach or the biological
role of the mutations introduced, the study
marks an important milestone in the devel-
opment and clinical application of gene-edi-
ted effector cell therapy.
Using recombinant Cas9 complexed with
four distinct single guide RNAs (sgRNAs),
the authors disrupted the TRAC, TRBC,
and PDCD1 genes with efciencies ranging
from 15% (for the TRBC gene) to 45% (for
the TRAC gene). The average lentiviral
NY-ESO1 TCR transduction efciency was
3.1%. Highlighting the inhibitory effect of
endogenous TCR and/or PD-1 expression
on the cytotoxicity of TCR-transgenic
T cells, disruption of these genes modestly
increased in vitro killing of tumor by
NYCE T cells. The selected sgRNAs had
minimal off-target activity, as evaluated us-
ing the iGUIDE method, and the identied
non-specic sites were not mapped to known
tumor suppressor genes or oncogenes in
T cells. Of note, chromosomal translocations
that arose from multiple simultaneous dou-
ble-strand breaks in CRISPR-edited T cells
were also detected in these patients. How-
ever, these genome alterations were unlikely
to confer survival or proliferative advantage
to the modied T cells, as the frequency of
both NY-ESO-1 TCR-positive and -negative
T cells carrying edited genes or transloca-
tions gradually decreased after infusion.
Notably, PD-1-decient T cells did not
expand or persist better than T cells with un-
altered PD-1 expression, consistent with
previous ndings that loss of PD-1 may
reduce the ability of T cells to establish a
long-lived memory population.
Upon infusion, NYCE T cells expanded and
tracked to tumor sites, stabilizing disease in
two of three patients treated on the protocol.
Although NYCE T cells remained detectable
in circulation for up to 9 months after infu-
sion, at least in one patient with myeloma,
the authors documented downregulation
of NY-ESO-1 and LAGE-1 antigens recog-
nized by the transgenic TCR. Evidence of
antigen escape, which has also been
frequently observed in studies using CAR
T cells for B cell malignancies,
contributed to the limited antitumor activity
of NYCE T cells and may warrant the use of
T cells simultaneously targeting multiple
tumor antigens.
Despite the presence of pre-existing anti-
bodies to the Cas9 protein from Strepto-
coccus pyogenes,
a component of the normal
commensal ora in humans, no reactivation
of Cas9-specic humoral immunity was
observed after administration of NYCE
T cells. The authors attributed this lack
of response to the minimal presence of
residual Cas9 protein in T cells at the time
of cryopreservation and the transient im-
mune suppression of patients induced by
the lymphodepleting treatment and prior
lymphotoxic therapies. While the authors
did not measure activation of endogenous
Cas9-specic T cells,
these responses, even
if present, did not blunt the expansion of
gene-edited T cells in vivo. Overall, these
results indicate genome editing of thera-
peutic T cells ex vivo using CRISPR/Cas9-
sgRNA RNP complexes results in minimal
additional immunogenicity, largely due to a
very transient persistence of electroporated
Cas9 protein in rapidly dividing T cells prior
to infusion.
Numerous proof-of-concept reports have
utilized genome-editing tools in T cells to
minimize the risk of graft-versus-host dis-
tumor immunosuppression,
T cell fratricide,
among other applica-
tions. Further, commercial and academic
entities have conducted several clinical
studies using gene-edited T cells, including
a landmark report in 2014 by Tebas et al.
utilizing zinc-nger nucleases to create
CCR5-edited T cells resistant to HIV infec-
tion. The latest study by Stadtmauer et al.
is the rst to report the manufacturing,
safety, and clinical activity of CRISPR/
Cas9-edited effector T cells.
Center for Cell and Gene Therapy, Baylor College of
Medicine, Houston Methodist Hospital and Texas
Childrens Hospital, Houston, TX, USA;
Program in Translational Biology and Molecular
Medicine, Baylor College of Medicine, Houston, TX,
Correspondence: Feiyan Mo, Center for Cell and
Gene Therapy, Baylor College of Medicine, Houston
Methodist Hospital and Texas Childrens Hospital,
Houston, TX, USA.
Molecular Therapy Vol. 28 No 4 April 2020 ª2020 The American Society of Gene and Cell Therapy. 995
Recent technological advances could further
improve the efciency and precision of
multiplexed genome editing and minimize
its side effects. Examples include employing
high-delity versions of Cas9 with reduced
off-target activity,
utilizing base editing
in lieu of non-homologous end-joining
(NHEJ) repair to minimize CRISPR-induced
double-strand breaks and the resulting
chromosomal translocations,
or knock-
ing in desired constructs to replace endoge-
nous genes via homology-directed recombi-
nation (HDR).
With the rst clinical
testing of CRISPR/Cas9-edited therapeutic
T cells, this pioneering study of NYCE
T cells is perhaps the end of the beginning
of the era of gene-edited immune effectors.
F.M., H.E.H., and M.M. wrote the
H.E.H. is a founder with equity in Allovir
and Marker Therapeutics; has served on
advisory boards for Gilead, Novartis, Tessa
Therapeutics, Kiadis, and PACT Pharma;
and receives research support from Tessa
Therapeutics and Kuur Therapeutics.
The National Cancer Institute
(P50CA126752), the SU2C/AACR (604817)
Meg Vosburg T cell Lymphoma Dream
Team, and the Leukemia and Lymphoma So-
ciety supported the authors.
1. Bailey, S.R., and Maus, M.V. (2019). Gene editing for
immune cell therapies. Nat. Biotechnol. 37, 1425
2. Stadtmauer, E.A., Fraietta, J.A., Davis, M.M., Cohen,
A.D., Weber, K.L., Lancaster, E., Mangan, P.A.,
Kulikovskaya, I., Gupta, M., Chen, F., Tian, L., et al.
(2020). CRISPR-engineered T cells in patients with
refractory cancer. Science 367, eaba7365.
3. Odorizzi, P.M., Pauken, K.E., Paley, M.A., Sharpe, A.,
and Wherry, E.J. (2015). Genetic absence of PD-1
promotes accumulation of terminally differentiated
exhausted CD8+ T cells. J. Exp. Med. 212, 11251137.
4. Majzner, R.G., and Mackall, C.L. (2018). Tumor anti-
gen escape from CAR T-cell therapy. Cancer Discov.
8, 12191226.
5. Charlesworth, C.T., Deshpande, P.S., Dever, D.P.,
Camarena, J., Lemgart, V.T., Cromer, M.K.,
Vakulskas, C.A., Collingwood, M.A., Zhang, L.,
Bode, N.M., et al. (2019). Identication of preexisting
adaptive immunity to Cas9 proteins in humans. Nat.
Med. 25, 249254.
6. Wagner, D.L., Amini, L., Wendering, D.J., Burkhardt,
L.M., Akyüz, L., Reinke, P., Volk, H.D., and
Schmueck-Henneresse, M. (2019). High prevalence
of Streptococcus pyogenes Cas9-reactive T cells
within the adult human population. Nat. Med. 25,
7. Poirot, L., Philip, B., Schiffer-Mannioui, C., Le Clerre,
D., Chion-Sotinel, I., Derniame, S., Potrel, P., Bas, C.,
Lemaire, L., Galetto, R., et al. (2015). Multiplex
Genome-Edited T-cell Manufacturing Platform for
Off-the-ShelfAdoptive T-cell Immunotherapies.
Cancer Res 75, 38533864.
8. Menger, L., Sledzinska, A., Bergerhoff, K., Vargas,
F.A., Smith, J., Poirot, L., Pule, M., Hererro, J.,
Peggs, K.S., and Quezada, S.A. (2016). TALEN-
Mediated Inactivation of PD-1 in Tumor-Reactive
Lymphocytes Promotes Intratumoral T-cell
Persistence and Rejection of Established Tumors.
Cancer Res. 76, 20872093.
9. Ren, J., Liu, X., Fang, C., Jiang, S., June, C.H., and
Zhao, Y. (2017). Multiplex Genome Editing to
Generate Universal CAR T Cells Resistant to PD1
Inhibition. Clin. Cancer Res. 23, 22552266.
10. Gomes-Silva, D., Srinivasan, M., Sharma, S., Lee,
C.M., Wagner, D.L., Davis, T.H., Rouce, R.H., Bao,
G., Brenner, M.K., and Mamonkin, M. (2017).
CD7-edited T cells expressing a CD7-specic CAR
for the therapy of T-cell malignancies. Blood 130,
11. Gomes-Silva, D., Atilla, E., Atilla, P.A., Mo, F.,
Tashiro, H., Srinivasan, M., Lulla, P., Rouce, R.H.,
Cabral, J.M.S., Ramos, C.A., et al. (2019). CD7 CAR
T Cells for the Therapy of Acute Myeloid Leukemia.
Mol. Ther. 27, 272280.
12. Tebas, P., Stein, D., Tang, W.W., Frank, I., Wang,
S.Q., Lee, G., Spratt, S.K., Surosky, R.T., Giedlin,
M.A., Nichol, G., et al. (2014). Gene editing of
CCR5 in autologous CD4 T cells of persons infected
with HIV. N. Engl. J. Med. 370, 901910.
13. Kleinstiver, B.P., Pattanayak, V., Prew, M.S., Tsai,
S.Q., Nguyen, N.T., Zheng, Z., and Joung, J.K.
(2016). High-delity CRISPR-Cas9 nucleases with
no detectable genome-wide off-target effects. Nature
529, 490495.
14. Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A., and
Liu, D.R. (2016). Programmable editing of a target
base in genomic DNA without double-stranded
DNA cleavage. Nature 533, 420424.
15. Anzalone, A.V., Randolph, P.B., Davis, J.R., Sousa,
A.A., Koblan, L.W., Levy, J.M., Chen, P.J., Wilson,
C., Newby, G.A., Raguram, A., and Liu, D.R. (2019).
Search-and-replace genome editing without double-
strand breaks or donor DNA. Nature 576, 149157.
16. Eyquem, J., Mansilla-Soto, J., Giavridis, T., van der
Stegen, S.J.C., Hamieh, M., Cunanan, K.M., Odak,
A., Gönen, M., and Sadelain, M. (2017). Targeting a
CAR to the TRAC locus with CRISPR/Cas9 enhances
tumour rejection. Nature 543, 113117.
996 Molecular Therapy Vol. 28 No 4 April 2020
... Furthermore, exciting new opportunities emerged thanks to gene editing/gene ablation techniques based on the revolutionary, highly specific and efficient CRISPR/Cas9 tools (332,333), which have been used not only to generate immunecheckpoint knock-outs (PD-1 KO) but also to design "universal" CARs, edited for TCR and/or HLA molecules expression (206,332,333), which could pave the road towards cost-effective allogeneic CAR-T cells for an "off-the-shelf" ACT with a broader spectrum (334,335). This technique can even be used for multiplexed genome editing (336). To this regard, feasibility of targeting multiple genes in T cells by multiplex CRISPR-Cas9 has recently been proven in a small interventional study in patients with advanced, refractory cancer (NCT03399448) (312). ...
... To this regard, feasibility of targeting multiple genes in T cells by multiplex CRISPR-Cas9 has recently been proven in a small interventional study in patients with advanced, refractory cancer (NCT03399448) (312). Further improvements of this technology are awaited as recent advances seem to insure increased precision and minimized side effects both in case of gene deletion and gene insertion (336). As allogeneic (allo)-CAR-T cells could offer readily available ACT sources that could expand the usage of CAR-cells based immunotherapy, other recent strategies for allo-CAR-T cells generation emerged, like the NKG2D (an NK-based activating receptor) expression. ...
Full-text available
During this last decade, adoptive transfer of T lymphocytes genetically modified to express chimeric antigen receptors (CARs) emerged as a valuable therapeutic strategy in hematological cancers. However, this immunotherapy has demonstrated limited efficacy in solid tumors. The main obstacle encountered by CAR-T cells in solid malignancies is the immunosuppressive tumor microenvironment (TME). The TME impedes tumor trafficking and penetration of T lymphocytes and installs an immunosuppressive milieu by producing suppressive soluble factors and by overexpressing negative immune checkpoints. In order to overcome these hurdles, new CAR-T cells engineering strategies were designed, to potentiate tumor recognition and infiltration and anti-cancer activity in the hostile TME. In this review, we provide an overview of the major mechanisms used by tumor cells to evade immune defenses and we critically expose the most optimistic engineering strategies to make CAR-T cell therapy a solid option for solid tumors.
... CRISPR technology is becoming the leading gene-editing tool, with increasingly expanding fields of application (30,31). These include HIV therapy, where excision of the integrated HIV-1 genome from cellular DNA can be achieved by taking advantage of CRISPR/Cas9 site-specific cleavage. ...
Full-text available
Gene editing may be used to excise the human immunodeficiency virus type-1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene-editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their re-transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of re-integration in a context of genomic instability, possibly caused by off-target activity of Cas9. IMPORTANCE Excision of HIV-1 provirus from host cell genome has proved feasible in vitro , and to some extent, in vivo. Among the different approaches, CRISPR/Cas9 is the most promising tool for gene editing. The present study underlines the remarkable effectiveness of CRISPR/Cas9 in removing the HIV-1 provirus from infected cells and investigates the fate of the excised HIV-1 genome. This study demonstrates that the free provirus may persist in the cell after editing and in appropriate circumstances may integrate back into the cell genome. As an episome, it might be transcriptionally active, especially in the presence of Tat and Rev. The persistence of HIV-1 episome was strongly decreased by gene editing with multiple targets. Although gene editing has the potential to eradicate HIV-1 infection, this work highlights a potential issue that warrants further investigation.
Hematopoietic stem cell transplantation (HSCT) has been used as a curative standard of care for moderate to severe primary immunodeficiency disorders as well as relapsed hematologic malignancies for over 50 years [1,2]. However, chronic and refractory viral infections remain a leading cause of morbidity and mortality in the immune deficient period following HSCT, where use of available antiviral pharmacotherapies is limited by toxicity and emerging resistance [3]. Adoptive immunotherapy using virus-specific T cells (VSTs) has been explored for over 2 decades [4,5] in patients post-HSCT and has been shown prior phase I-II studies to be safe and effective for treatment or preventions of viral infections including cytomegalovirus, Epstein-Barr virus, BK virus, and adenovirus with minimal toxicity and low risk of graft vs host disease [6-9]. This review summarizes methodologies to generate VSTs the clinical results utilizing VST therapeutics and the challenges and future directions for the field.
Full-text available
CRISPR takes first steps in humans CRISPR-Cas9 is a revolutionary gene-editing technology that offers the potential to treat diseases such as cancer, but the effects of CRISPR in patients are currently unknown. Stadtmauer et al. report a phase 1 clinical trial to assess the safety and feasibility of CRISPR-Cas9 gene editing in three patients with advanced cancer (see the Perspective by Hamilton and Doudna). They removed immune cells called T lymphocytes from patients and used CRISPR-Cas9 to disrupt three genes ( TRAC, TRBC , and PDCD1 ) with the goal of improving antitumor immunity. A cancer-targeting transgene, NY-ESO-1, was also introduced to recognize tumors. The engineered cells were administered to patients and were well tolerated, with durable engraftment observed for the study duration. These encouraging observations pave the way for future trials to study CRISPR-engineered cancer immunotherapies. Science , this issue p. eaba7365 ; see also p. 976
Full-text available
Most genetic variants that contribute to disease¹ are challenging to correct efficiently and without excess byproducts2–5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells including targeted insertions, deletions, and all 12 types of point mutation without requiring double-strand breaks or donor DNA templates. We applied prime editing in human cells to correct efficiently and with few byproducts the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA), to install a protective transversion in PRNP, and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing offers efficiency and product purity advantages over homology-directed repair, complementary strengths and weaknesses compared to base editing, and much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct about 89% of known pathogenic human genetic variants.
Full-text available
The CRISPR–Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR–Cas9 system to therapeutically correct human genetic diseases1–6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes5,7. Given that these two bacterial species infect the human population at high frequencies8,9, we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR–Cas9 system moves toward clinical trials.
Full-text available
The discovery of the highly efficient site-specific nuclease system CRISPR-Cas9 from Streptococcus pyogenes has galvanized the field of gene therapy. The immunogenicity of Cas9 nuclease has been demonstrated in mice. Preexisting immunity against therapeutic gene vectors or their cargo can decrease the efficacy of a potentially curative treatment and may pose significant safety issues. S. pyogenes is a common cause for infectious diseases in humans, but it remains unclear whether it induces a T cell memory against the Cas9 nuclease. Here, we show the presence of a preexisting ubiquitous effector T cell response directed toward the most widely used Cas9 homolog from S. pyogenes (SpCas9) within healthy humans. We characterize SpCas9-reactive T cells within the CD4/CD8 compartments for multi-effector potency, cytotoxicity, and lineage determination. In-depth analysis of SpCas9-reactive T cells reveals a high frequency of SpCas9-reactive regulatory T cells that can mitigate SpCas9-reactive effector T cell proliferation and function in vitro. Our results shed light on T cell-mediated immunity toward CRISPR-associated nucleases and offer a possible solution to overcome the problem of preexisting immunity.
Full-text available
Chimeric antigen receptor (CAR) T cell therapy for the treatment of acute myeloid leukemia (AML) has the risk of toxicity to normal myeloid cells. CD7 is expressed by the leukemic blasts and malignant progenitor cells of approximately 30% of AML patients but is absent on normal myeloid and erythroid cells. Since CD7 expression by malignant blasts is also linked with chemoresistance and poor outcomes, targeting this antigen may be beneficial for this subset of AML patients. Here, we show that expression of a CD7-directed CAR in CD7 gene-edited (CD7KO) T cells effectively eliminates CD7⁺ AML cell lines, primary CD7⁺ AML, and colony-forming cells but spares myeloid and erythroid progenitor cells and their progeny. In a xenograft model, CD7 CAR T cells protect mice against systemic leukemia, prolonging survival. Our results support the feasibility of using CD7KO CD7 CAR T cells for the non-myeloablative treatment of CD7⁺ AML. CD7 is expressed in T cells and in some subtypes of AML. Gomes-Silva and colleagues show that CRISPR-edited CD7 knockout T cells expressing a CD7-specific CAR resist fratricide and control AML while sparing the non-malignant myeloid compartment.
Full-text available
Key Points Genomic disruption of CD7 prior to CAR transduction allows generation of CD7 CAR T cells without extensive self-antigen-driven fratricide. CD7 CAR T cells have robust activity against T-cell malignancies in vitro and in vivo.
Full-text available
CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations at genomic locations that resemble the intended target. These so-called off-target effects can confound research applications and are important considerations for potential therapeutic use. Existing strategies for reducing genome-wide off-targets of the broadly used Streptococcus pyogenes Cas9 (SpCas9) have thus far proven to be imperfect by possessing only partial efficacy and/or other limitations that constrain their use.
Autologous T cells that have been genetically modified to express a chimeric antigen receptor (CAR) targeting the B cell antigen CD19 have yielded remarkable clinical responses in patients with B cell malignancies, and are now on the market as anticancer ‘drugs’. Riding on this success, the field of immune cell engineering is rapidly growing, with creative solutions to major outstanding challenges, such as limitations in target antigen selection, the hostility of the tumor microenvironment and the logistical challenges of generating autologous therapies. Innovations in antigen receptor design, coupled with advances in gene transfer and gene-editing technologies, have enabled the engineering of T cells to have sophisticated sensing circuits, to have synthetic functionalities, and to be used as off-the-shelf, universal cellular products. As these technologies are applied to other immune cells, such as natural killer cells, hematopoietic cells or induced pluripotent stem cells, the potential to transform the treatment of many cancers, as well as other diseases, is palpably exciting. We discuss the pipeline of several influential innovations in the preclinical setting, the early translational results from clinical trials of these next-generation approaches, and the outlook for gene-modified or gene-edited cell therapies. Bailey and Maus discuss cutting-edge developments in engineering immune cells towards expanding the reach and efficacy of adoptive cell therapies in cancer and beyond.
Emerging data from chimeric antigen receptor (CAR) T-cell trials in B-cell malignancies demonstrate that a common mechanism of resistance to this novel class of therapeutics is the emergence of tumors with loss or downregulation of the target antigen. Antigen loss or antigen-low escape is likely to emerge as an even greater barrier to success in solid tumors, which manifest greater heterogeneity in target antigen expression. Potential approaches to overcome this challenge include engineering CAR T cells to achieve multispecificity and to respond to lower levels of target antigen and more efficient induction of natural antitumor immune responses as a result of CAR-induced inflammation. In this article, we review the evidence to date for antigen escape and downregulation and discuss approaches currently under study to overcome these obstacles. Significance: Antigen escape and downregulation have emerged as major issues impacting the durability of CAR T-cell therapy. Here, we explore their incidence and ways to overcome these obstacles in order to improve clinical outcomes.
Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumour rejection. The most successful CARs used to date are those targeting CD19 (ref. 2), which offer the prospect of complete remission in patients with chemorefractory or relapsed B-cell malignancies. CARs are typically transduced into the T cells of a patient using γ-retroviral vectors or other randomly integrating vectors, which may result in clonal expansion, oncogenic transformation, variegated transgene expression and transcriptional silencing. Recent advances in genome editing enable efficient sequence-specific interventions in human cells, including targeted gene delivery to the CCR5 and AAVS1 loci. Here we show that directing a CD19-specific CAR to the T-cell receptor α constant (TRAC) locus not only results in uniform CAR expression in human peripheral blood T cells, but also enhances T-cell potency, with edited cells vastly outperforming conventionally generated CAR T cells in a mouse model of acute lymphoblastic leukaemia. We further demonstrate that targeting the CAR to the TRAC locus averts tonic CAR signalling and establishes effective internalization and re-expression of the CAR following single or repeated exposure to antigen, delaying effector T-cell differentiation and exhaustion. These findings uncover facets of CAR immunobiology and underscore the potential of CRISPR/Cas9 genome editing to advance immunotherapies.