Biology subjects

Roth, T. L.

Publications and source records attributed to Roth, T. L..

18 recordsLinked to original sources

bioRxiv2026

Ultra-large targeted DNA integrations in primary human cells

Genetic engineering experiments and therapies are constrained by the size of DNA integrations into human cells genomes. Existing AAV, lentiviral, and non-viral methods rapidly decrease in integration efficiency beyond [~]5kb of sequence. Through systematic evaluation of non-viral DNA template formats, we identified circular ssDNA and dsDNA as capable of mediating >5kb integrations. Large circular DNA delivery efficiency and its impacts on cell viability and payload expression could be significantly improved with small DNA "helper" plasmids, mRNA-encoded nucleases, and sequence design optimizations. Collectively, these modifications enabled ultra-large--up to 10 kb DNA--integrations at >20% efficiency in primary human T cells at the TRAC locus and at >60% efficiency in human iPSCs at the AAVS1 locus. Finally, we demonstrate that GMP clinical-manufactured T cells with ultra-large integrations are functional in vitro and in vivo. Overall, we identified optimal template architectures, delivery modes, and sequence design rules for ultra-large DNA integrations in both research and clinical settings to accelerate basic genetic research and next-generation cellular therapies.

genetics↗

bioRxiv2026

Systematic identification of seed-driven off-target effects in Perturb-seq experiments

Genome-wide Perturb-seq (GWPS) has emerged as a powerful approach for unbiased mapping of gene regulatory networks. A key assumption underlying many Perturb-seq analyses is that each guide RNA exclusively perturbs a single target locus. Without methods to identify and filter off-target events, erroneous gene-pathway associations driven by off-target activity can propagate into downstream analyses. Here, we present a workflow for the systematic identification of candidate off-target events in CRISPRi Perturb-seq experiments. Our approach exploits the observation that cells harboring a guide which represses an off-target gene display transcriptional similarity to cells in which that gene is directly targeted by an on-target guide. We apply our workflow to multiple GWPS datasets and nominate off-target events in which a guide nominally targeting one gene also represses a distinct gene producing a phenotype likely attributable to the off-target perturbation. We use both off-target gene repression and guide seed sequence alignments at the off-target promoter locus as evidence for off-target effects and find independent evidence of putative off-target events in separate GWPS datasets. Together, these results establish a principled framework for the identification and filtering of off-target guide effects in Perturb-seq experiments.

genomics↗

bioRxiv2026

Molecular pixelation of the CAR T cell surface proteome

Immunotherapies using CAR T cells are revolutionizing B-cell acute lymphoblastic leukemia treatments. However, the majority of patients remain unresponsive, and chronic stimulation of T cells is a common contributor that reduces effector function and persistence. We apply Molecular Pixelation, a recently developed single-cell technology for characterizing cellular surface proteomes, to determine characteristic topological surface-based proteomic signatures of CAR T cell exhaustion. We analyze 76 surface proteins on 8504 CAR T cells at a single-cell level, collected from three donors and either stimulated once or repeatedly, six times over two weeks. The abundances, polarizations, and colocalizations of surface proteins can each distinguish CAR T cells that were stimulated acutely or chronically, and all but one marker with polarization changes increased in polarization. These data also reveal disrupted adhesion signatures of protein colocalization in the peripheral supramolecular activation complex (pSMAC) and increased CD37/CD82 colocalization after chronic stimulation. These Molecular Pixelation results convey new spatial signatures for proteomic polarization and colocalization on the cell surface that represent new cell-state axes for immunology and systems biology.

systems biology↗

bioRxiv2025

A unified genetic perturbation language for human cellular programming

Evolution simultaneously and combinatorially explores complex genetic changes across perturbation classes, including gene knockouts, knockdowns, overexpression, and the creation of new genes from existing domains. Separate technologies are capable of genetic perturbations at scale in human cells, but these methods are largely mutually incompatible. Here we present CRISPR-All, a unified genetic perturbation language for programming of any major type of genetic perturbation simultaneously, in any combination, at genome scale, in primary human cells. This is enabled by a standardized molecular architecture for each major perturbation class, development of a functional syntax for combining arbitrary numbers of elements across classes, and linkage to unique single cell compatible barcodes. To facilitate use, CRISPR-All converts high level descriptions of desired complex genetic changes into a single DNA sequence that can rewire genomic programs within a cell. Using the CRISPR-All language allowed for head-to-head functional comparisons across perturbation types in a comprehensive analysis of all previously identified genetic enhancements of human CAR-T cells. Combining CRISPR-All programs with single cell RNA sequencing revealed a greater diversity of phenotypic states, including improved functional performance, only accessible through distinct perturbation classes. Finally, CRISPR-All combinatorial genome scale screening of up to four distinct perturbations simultaneously revealed additive functional improvements in human T cells accessible only through iterative multiplexing of modifications across perturbation classes. CRISPR-All enables exploration of a combinatorial genetic perturbation space, which may be impactful for biological and clinical applications.

genetics↗

bioRxiv2025

Scalable transcription factor mapping uncovers the regulatory dynamics of natural and synthetic transcription factors in human T cell states

Heterogeneous T cell states are critical in immune responses and have been explored by CRISPR-based and synthetic domain-swapped transcription factor (TF) screens, yielding novel insights and immunotherapeutics. However, a scalable strategy to map TFs in primary human T cells is lacking, which limits our understanding of the functions of critical TFs. We therefore adapted a transposon-based TF mapping strategy termed Calling Cards for primary human CD8 T cells, applying it to five key TFs with undefined binding sites in this cell type: TOX, TOX2, TCF7, SOX4, and RBPJ. To derive biological insights from these data, we developed an analytical framework to integrate TF binding with multi-omic sequencing data, revealing convergence of TOX and TCF7 binding at dynamic enhancers of memory CD8 T cells. We then identified TF co-bound gene programs related to memory and exhaustion states in addition to putative gene targets of known and unappreciated TF roles, including TOX binding at critical genes of both exhaustion and terminal effector memory differentiation. To further scale our TF analysis platform, we modified Calling Cards to create TFlex: a method uniquely suited for multiplexed mapping of paralogous TFs. We applied TFlex to simultaneously map eight natural and domain-swapped TFs in primary human CD8 T cells, which demonstrated that domain-swapped TFs display emergent behavior in binding site selection and transcriptional effects on target genes that cannot be estimated as the sum of their constituent domains. Collectively, our data highlight the importance of scalable TF mapping in primary human T cells to elucidate TF function and the transcriptional regulation of cell states.

immunology↗

bioRxiv2024

Evolutionarily guided transcription factor design programs novel T cell states

Protein-coding genes in the human genome evolved via modular rearrangement of domains from ancestral genes1. Here, we develop a scalable, evolutionarily guided method to assemble novel protein-coding genes from constituent domains within a protein family, termed DESynR (Domain Engineered via Synthesis and Recombination) genes. Using primary human chimeric antigen receptor T cells as a model system, we find that the expression of DESynR Activator Protein-1 (AP-1) transcription factors (TFs) significantly outperforms the overexpression of natural AP-1 TFs in multiple functional assays in vitro and in vivo. Top DESynR AP-1 TFs exhibit non-intuitive architectures of constituent domains, including from TFs that are not canonically expressed in T cells. DESynR AP-1 TFs induce broad transcriptional and epigenetic reprogramming of T cells and, in some cases, lead to the development of non-natural T cell states, engaging gene expression modules from disparate human cell types. Taken together, we demonstrate that novel configurations of existing protein domains may uncover non-evolved genes that program cell states with therapeutically relevant functions.

genetics↗

bioRxiv2024

Non-viral Intron Knockins Enable Simplified and Flexible Targeting of Endogenous Genes

Targeting new genetic material to endogenous genes has opened diverse therapeutic and research applications, but current exon-based targeting methods have limited integration sites and are compatible only with complex or harsh selection methods. We present non-viral intron targeting, integrating large synthetic exons into endogenous introns to increase targeting flexibility and simplify selection of successfully edited cells. Engineered control of large synthetic exons splicing behavior further generalizes cell and gene therapy applications of non-viral intron knockins.

bioengineering↗

bioRxiv2022

Latent human herpesvirus 6 is reactivated in chimeric antigen receptor T cells

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are still being understood. For example, we currently lack a comprehensive understanding of the mechanisms of neurotoxicity observed in patients receiving T cell therapies, including recent reports of encephalitis caused by human herpesvirus 6 (HHV-6) reactivation1. Here, via petabase-scale viral RNA data mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in human CD4+ T cells in standard in vitro cultures. Using single-cell sequencing, we identify a rare population of HHV-6 super-expressors (~1 in 300-10,000 cells) that possess high viral transcriptional activity in chimeric antigen receptor (CAR) T cell culture before spreading to infect other cells in vitro. Through the analysis of single-cell sequencing data from patients receiving cell therapy products that are FDA-approved2 or used in clinical studies3,4, we identify the presence of CAR+, HHV-6 super-expressor T cells in vivo. Together, our study implicates cell therapy products as a source of lytic HHV-6 reported in clinical trials1,5-7 and has broad implications for the design, production, and monitoring of cell therapies.

immunology↗

bioRxiv2022

Modular Pooled Discovery of Synthetic Knockin Sequences to Program Durable Cell Therapies

Chronic stimulation can cause T cell dysfunction and limit efficacy of cellular immunotherapies. CRISPR screens have nominated gene targets for engineered T cells, but improved methods are required to compare large numbers of synthetic knockin sequences to reprogram cell functions. Here, we developed Modular Pooled Knockin Screening (ModPoKI), an adaptable platform for modular construction of DNA knockin libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors. Over 20 ModPoKI screens across human TCR and CAR T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct to enhance long-term T cell fitness and anti-cancer function in vitro and in vivo. ModPoKIs modularity allowed us to generate a [~]10,000-member library of TF combinations. Non-viral knockin of a combined BATF-TFAP4 polycistronic construct further enhanced function in vivo. ModPoKI facilitates discovery of complex gene constructs to program cellular functions. HighlightsO_LIModular pooled knockins of hundreds of TF and surface receptor constructs combined with different antigen receptors C_LIO_LIChronic stimulation screens discover programs to improve T cell persistence C_LIO_LICombinatorial knockin screens with [~]10,000 transcription factor combinations C_LIO_LIBATF-TFAP4 dual knockin construct improves CAR T cell function in vitro and in vivo C_LI

immunology↗

bioRxiv2021

Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing

CRISPR-Cas9 offers unprecedented opportunities to modify genome sequences in primary human cells to study disease variants and reprogram cell functions for next-generation cellular therapies. CRISPR has several potential advantages over widely used retroviral vectors including: 1) site-specific transgene insertion via homology directed repair (HDR), and 2) reductions in the cost and complexity of genome modification. Despite rapid progress with ex vivo CRISPR genome engineering, many novel research and clinical applications would be enabled by methods to further improve knock-in efficiency and the absolute yield of live knock-in cells, especially with large HDR templates (HDRT). We recently reported that Cas9 target sequences (CTS) could be introduced into double-stranded DNA (dsDNA) HDRTs to improve knock-in, but yields and efficiencies were limited by toxicity at high HDRT concentrations. Here we developed a novel system that takes advantage of lower toxicity with single-stranded DNA (ssDNA). We designed hybrid ssDNA HDRTs that incorporate CTS sites and were able to boost knock-in percentages by >5-fold and live cell yields by >7-fold relative to dsDNA HDRTs with CTS. Knock-in efficiency and yield with ssCTS HDRTs were increased further with small molecule inhibitor combinations to improve HDR. We demonstrate application of these methods across a variety of target loci, knock-in constructs, and primary human cell types to reach ultra-high HDR efficiencies (>80-90%) which we use for pathogenic gene variant modeling and universal gene replacement strategies for IL2RA and CTLA4 mutations associated with mendelian immune disorders. Finally, we develop a GMP-compatible method for fully non-viral CAR-T cell manufacturing, demonstrating knock-in efficiencies of 46-62% and generating yields of >1.5 x 109 CAR+ T cells, well above current doses for adoptive cellular therapies. Taken together, we present a comprehensive non-viral approach to model disease associated mutations and re-write targeted genome sequences to program immune cell therapies at a scale compatible with future clinical application.

synthetic biology↗

bioRxiv2020

Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins

Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically-edited cells that retain critical markers of both myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection more than fifty-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate development of novel myeloid cellular therapies.

genetics↗

bioRxiv2020

TCF-1 regulates the stem-like memory potential of HIV-specific CD8+ T cells in elite controllers

Although many HIV cure strategies seek to expand HIV-specific CD8+ T cells to control the virus, all are likely to fail if cellular exhaustion is not prevented. A loss in stem-like memory properties (i.e., the ability to proliferate and generate secondary effector cells) is a key feature of exhaustion; little is known, however, about how these properties are regulated in human virus-specific CD8+ T cells. We found that virus-specific CD8+ T cells from humans and non-human primates naturally controlling HIV/SIV infection express more of the transcription factor, TCF-1, than non-controllers. HIV-specific CD8+ T cell TCF-1 expression correlated with memory marker expression and proliferative capacity and declined with antigenic stimulation. CRISPR-Cas9 editing of TCF-1 in human primary T cells demonstrated a direct role in regulating expansion capacity. Collectively, these data suggest that TCF-1 controls the stem-like memory properties of HIV-specific CD8+ T cells and provides a rationale for enhancing this pathway in T cell-based therapeutic strategies for HIV. One Sentence SummaryTCF-1 is highly expressed in HIV-specific CD8+ T cells from elite controllers and directly regulates human CD8+ T cell expansion capacity in response to T cell receptor stimulation.

immunology↗

bioRxiv2019

Dynamics of T cell activation mediated by eIF3 interactions with T cell receptor mRNAs

Activation of T cells requires a global surge in cellular protein synthesis, accompanied by a large increase in translation initiation1-4. A central component of the translation initiation machinery-the multi-subunit eukaryotic initiation factor 3 (eIF3)-is rapidly turned on when quiescent T cells are stimulated3. However, the precise role eIF3 plays in activated T cells is not known. Using a global transcriptome crosslinking approach, we show human eIF3 interacts with a distinct set of mRNAs in activated Jurkat cells. A subset of these mRNAs, including those encoding the T cell receptor (TCR) subunits TCRA and TCRB, crosslink to eIF3 across the entire length of the mRNA. The TCRA and TCRB mRNAs do not co-localize with translationally repressed environments of P-bodies or stress granules but form distinct granules, potentially acting as translation "hot-spots." T cell activation through CD28 causes a burst of TCR translation controlled by elements in the 3-untranslated regions (3-UTRs) of the TCRA and TCRB mRNAs that directly contact eIF3 and that are required for T cell activity. These results highlight a new role for eIF3 in regulating the translation dynamics of the TCR and provide insights that can guide the engineering of T cells used in cell immunotherapy applications.

molecular biology↗

bioRxiv2019

Rapid discovery of synthetic DNA sequences to rewrite endogenous T cell circuits

Genetically-engineered immune cell therapies have been in development for decades1-3 and recently have proven effective to treat some types of cancer4. CRISPR-based genome editing methods, enabling more flexible and targeted sequence integrations than viral transduction, have the potential to extend the clinical utility of cell therapies5,6. Realization of this potential depends on improved knowledge of how coding and non-coding sites throughout the genome can be modified efficiently and on improved methods to discover novel synthetic DNA sequences that can be introduced at targeted sites to enhance critical immune cell functions. Here, we developed improved guidelines for non-viral genome targeting in human T cells and a pooled discovery platform to identify synthetic genome modifications that enhance therapeutically-relevant cell functions. We demonstrated the breadth of targetable genomic loci by performing large knock-ins at 91 different genomic sites in primary human T cells, and established the power of flexible genome targeting by generating cells with Genetically Engineered Endogenous Proteins (GEEPs) that seamlessly integrate synthetic and endogenous genetic elements to alter signaling input, output, or regulatory control of genes encoding key immune receptors. Motivated by success in introducing synthetic circuits into endogenous sites, we then developed a platform to facilitate discovery of novel multi-gene sequences that reprogram both T cell specificity and function. We knocked in barcoded pools of large DNA sequences encoding polycistronic gene programs. High-throughput pooled screening of targeted knock-ins to the endogenous T cell receptor (TCR) locus revealed a transcriptional regulator and novel protein chimeras that combined with a new TCR specificity to enhance T cell responses in the presence of suppressive conditions in vitro and in vivo. Overall, these pre-clinical studies provide flexible tools to discover complex synthetic gene programs that can be written into targeted genome sites to generate more effective therapeutic cells.

immunology↗

bioRxiv2019

A Cas9 nanoparticle system with truncated Cas9 target sequences on DNA repair templates enhances genome targeting in diverse human immune cell types

Virus-modified T cells are approved for cancer immunotherapy, but more versatile and precise genome modifications are needed for a wider range of adoptive cellular therapies1-4. We recently developed a non-viral CRISPR-Cas9 system for genomic site-specific integration of large DNA sequences in primary human T cells5. Here, we report two key improvements for efficiency and viability in an expanded variety of clinically-relevant primary cell types. We discovered that addition of truncated Cas9 target sequences (tCTS) at the ends of the homology directed repair (HDR) templates can interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template and enhance targeting efficiency. Further, stabilizing the Cas9 RNPs into nanoparticles with poly(glutamic acid) improved editing, reduced toxicity, and enabled lyophilized storage without loss of activity. Combining the tCTS HDR template modifications with polymer-stabilized nanoparticles increased gene targeting efficiency and viable cell yield across multiple genomic loci in diverse cell types. This system is an inexpensive, user-friendly delivery platform for non-viral genome reprogramming that we successfully applied in regulatory T cells (Tregs), {gamma}{delta}-T cells, B cells, NK cells, and primary and iPS-derived6 hematopoietic stem progenitor cells (HSPCs).

bioengineering↗

bioRxiv2018

Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function

Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, sgRNA lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes validated hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA-Seq revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling response to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.

immunology↗

bioRxiv2017

A CRISPR-Cas9 Genome Engineering Platform in Primary CD4+ T Cells for the Interrogation of HIV Host Factors

CRISPR-Cas9 gene editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology to primary human T cells to generate a high-throughput platform for analyzing the role of host factors in pathogen infection and lifecycle. Here, we describe applications of this system to investigate HIV pathogenesis in CD4+ T cells. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated primary human CD4+ T cells by nucleofection. These edited cells are then validated and expanded for use in downstream cellular, genetic, or protein-based assays. Our platform supports the arrayed generation of several gene manipulations in only a few hours time and is widely adaptable across culture conditions, infection protocols, and downstream applications. We present detailed protocols for crRNP synthesis, primary T cell culture, 96-well nucleofection, molecular validation, and HIV infection with additional considerations for guide and screen design as well as crRNP multiplexing.

systems biology↗

bioRxiv2017

Correction of autoimmune IL2RA mutations in primary human T cells using non-viral genome targeting

Human T cells are central to physiological immune homeostasis, which protects us from pathogens without collateral autoimmune inflammation. They are also the main effectors in most current cancer immunotherapy strategies1. Several decades of work have aimed to genetically reprogram T cells for therapeutic purposes2-5, but as human T cells are resistant to most standard methods of large DNA insertion these approaches have relied on recombinant viral vectors, which do not target transgenes to specific genomic sites6, 7. In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homology-directed repair (HDR), but to date in human T cells this still requires viral transduction8, 9. Here, we developed a non-viral, CRISPR-Cas9 genome targeting system that permits the rapid and efficient insertion of individual or multiplexed large (>1 kilobase) DNA sequences at specific sites in the genomes of primary human T cells while preserving cell viability and function. We successfully tested the potential therapeutic use of this approach in two settings. First, we corrected a pathogenic IL2RA mutation in primary T cells from multiple family members with monogenic autoimmune disease and demonstrated enhanced signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized the tumour antigen, with concomitant cytokine release and tumour cell killing. Taken together, these studies provide preclinical evidence that non-viral genome targeting will enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

genetics↗