Dennis James Zhang

All Publications

• Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells NATURE COMMUNICATIONS Lampe, G. D., Liang, A. R., Zhang, D. J., Fernandez, I. S., Sternberg, S. H. 2025; 16 (1): 7891

  Abstract

  Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in heterogeneous mixtures of undesirable outcomes. We recently leveraged a type I-F CRISPR-associated transposase, PseCAST, for DSB-free DNA integration in human cells, albeit at low efficiencies; multiple lines of evidence suggest DNA binding may be a bottleneck for higher efficiencies. Here we report structural determinants of DNA recognition by the PseCAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), revealing subtype-specific interactions and RNA-DNA heteroduplex features. By combining structural data, library screens, and rationally engineered mutants, we uncover variants with increased integration efficiencies and modified PAM stringencies. We further leverage transpososome structural predictions to build hybrid CASTs that combine orthogonal DNA binding and integration modules. Our work provides unique structural insights into type I-F CASTs and showcases diverse strategies to investigate and engineer RNA-guided transposase architectures for human genome editing applications.

  View details for DOI 10.1038/s41467-025-63164-0

  View details for Web of Science ID 001559616000025

  View details for PubMedID 40849322

  View details for PubMedCentralID PMC12375015

• Protein-primed homopolymer synthesis by an antiviral reverse transcriptase NATURE Tang, S., Zedaveinyte, R., Burman, N., Pandey, S., Ramirez, J. L., Kulber, L. M., Wiegand, T., Wilkinson, R. A., Ma, Y., Zhang, D. J., Lampe, G. D., Berisa, M., Jovanovic, M., Wiedenheft, B., Sternberg, S. H. 2025; 643 (8074): 1352-1362

  Abstract

  Bacteria defend themselves from viral predation using diverse immune systems, many of which target foreign DNA for degradation1. Defence-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this strategy by using DNA synthesis instead2,3. We and others recently showed that DRT2 systems use an RNA template to assemble a de novo gene that encodes the antiviral effector protein Neo4,5. It remains unclear whether similar mechanisms of defence are used by other related DRT families. Here, we show that DRT9 systems defend against phage using DNA homopolymer synthesis. Viral infection triggers polydeoxyadenylate (poly-dA) accumulation in the cell, driving abortive infection and population-level immunity. Cryo-electron microscopy structures reveal how a non-coding RNA serves as both a structural scaffold and reverse transcription template to direct hexameric complex assembly and poly-dA synthesis. Notably, biochemical and functional experiments identify tyrosine residues within the reverse transcriptase itself that probably prime DNA synthesis, leading to the formation of protein-DNA covalent adducts. Synthesis of poly-dA by DRT9 in vivo is regulated by the competing activities of phage-encoded triggers and host-encoded silencers. Collectively, our study identifies a nucleic-acid-driven defence system that expands the paradigm of bacterial immunity and broadens the known functions of reverse transcriptases.

  View details for DOI 10.1038/s41586-025-09179-5

  View details for Web of Science ID 001516522000001

  View details for PubMedID 40436039

  View details for PubMedCentralID PMC12483538

• De novo gene synthesis by an antiviral reverse transcriptase SCIENCE Tang, S., Conte, V., Zhang, D. J., Zedaveinyte, R., Lampe, G. D., Wiegand, T., Tang, L. C., Wang, M., Walker, M. W. G., George, J., Berchowitz, L. E., Jovanovic, M., Sternberg, S. H. 2024; 386 (6717): eadq0876

  Abstract

  Defense-associated reverse transcriptase (DRT) systems perform DNA synthesis to protect bacteria against viral infection, but the identities and functions of their DNA products remain largely unknown. We show that DRT2 systems encode an unprecedented immune pathway that involves de novo gene synthesis through rolling circle reverse transcription of a noncoding RNA (ncRNA). Programmed template jumping on the ncRNA generates a concatemeric cDNA, which becomes double-stranded upon viral infection. This DNA product constitutes a protein-coding, nearly endless open reading frame (neo) gene whose expression leads to potent cell growth arrest, restricting the viral infection. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA.

  View details for DOI 10.1126/science.adq0876

  View details for Web of Science ID 001386690800027

  View details for PubMedID 39116258

  View details for PubMedCentralID PMC11758365

• Novel molecular requirements for CRISPR RNA-guided transposition NUCLEIC ACIDS RESEARCH Walker, M. W. G., Klompe, S. E., Zhang, D. J., Sternberg, S. H. 2023; 51 (9): 4519-4535

  Abstract

  CRISPR-associated transposases (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system (VchCAST). On the donor DNA, large transposon end libraries revealed binding site nucleotide preferences for the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications with CAST systems.

  View details for DOI 10.1093/nar/gkad270

  View details for Web of Science ID 000971451900001

  View details for PubMedID 37078593

  View details for PubMedCentralID PMC10201428

• Coordinated synthesis of double-stranded DNA by a dual reverse transcriptase immune system. bioRxiv : the preprint server for biology Wang, M., Yoneyama, K., Zedaveinyte, R., Ishikawa, J., Tang, S., Le, H. C., Wiegand, T., Ramirez, J. L., Nagahata, N., Ma, Y., Zhang, D. J., Helmeczi, E., Berisa, M., Jovanovic, M., Hiraizumi, M., Yamashita, K., Nishimasu, H., Sternberg, S. H. 2026

  Abstract

  Recent studies have revealed that defense-associated reverse transcriptase (DRT) systems mediate antiviral immunity through distinct modes of cDNA synthesis. Class I DRTs catalyze untemplated DNA synthesis with random or nucleotide-biased sequences, whereas Class II DRTs polymerize noncoding RNA-templated products, including concatemeric repeats and homopolymeric cDNA. However, how these distinct modes of cDNA synthesis are employed to drive antiviral defense remains poorly understood. Here, we report an unprecedented mechanism of DRT3 immunity, in which RT enzymes from both Class I and Class II coordinate their diverse activities to produce self-complementary double-stranded DNA (dsDNA). Remarkably, whereas the DRT3a enzyme relies on a 5'-ACACAC-3' RNA template to synthesize long poly-(dTdG) repeats, DRT3b synthesizes precise poly-(dCdA) repeats without any nucleic acid template at all. Cryo-electron microscopy structures reveal that DRT3b assembles into a hexameric complex and employs active site-adjacent residues to function as deoxyadenosine and deoxycytidine gates that enforce alternating addition to produce dinucleotide repeats, representing a unique example of amino acid-templated DNA polymerization. Strikingly, DRT3 immune systems are toxic in a genetic background lacking E. coli RecBCD, implicating host recombination machinery in limiting DRT3-mediated dsDNA levels. Consistent with this model, we discovered that the phage-encoded RecBCD inhibitor, Gam, potently triggers DRT3-mediated abortive infection. Collectively, our findings reveal how two polymerases with distinct templating strategies cooperate to generate complementary DNA and drive antiviral defense.

  View details for DOI 10.64898/2026.05.04.722794

  View details for PubMedID 42146563

• Antiviral reverse transcriptases reveal the evolutionary origin of telomerase. bioRxiv : the preprint server for biology Tang, S., Ramirez, J. L., Mestre, M. R., Zhang, D. J., Wang, M., Wiegand, T., Ma, Y., Jovanovic, M., Pinilla-Redondo, R., Sternberg, S. H. 2025

  Abstract

  Defense-associated reverse transcriptases (DRTs) employ diverse and distinctive mechanisms of cDNA synthesis to protect bacteria against viral infection. However, much of DRT family diversity remains unstudied. Here we identify a new antiviral defense system, DRT10, that associates with a noncoding RNA (ncRNA) to catalyze processive, protein-primed synthesis of tandem-repeat DNA. Repeat addition is dictated by sequence and structural features of the ncRNA that have direct parallels in the RNA component of telomerase. Remarkably, a phylogenetic analysis of RTs across domains of life reveals an unexpected link between DRT10 and telomerase that is further supported by structural comparisons and mechanistic evidence. These findings expand the repertoire of reverse transcription mechanisms in antiviral defense and point to a bacterial origin for telomerase.

  View details for DOI 10.1101/2025.10.16.682844

  View details for PubMedID 41279515

• The All of Us Research Program's Social Media Outreach to Underrepresented Populations: Mixed Methods Analysis. Journal of medical Internet research Zhang, D. J., Bentz, M. G., Shim, J. K., Lee, S. S. 2025; 27: e63793

  Abstract

  The All of Us Research Program (AoURP) is a prominent precision medicine research initiative committed to diverse participation. The program harnesses digital outreach as a key strategy for recruiting and retaining underrepresented populations, using language that sometimes invokes notions of solidarity. This targeted recruitment of underrepresented groups and potential use of solidaristic language raise concerns about how participation will manifest tangible benefits for these populations and whether institutions assume responsibility for addressing past and present research harms.This study examines how the AoURP conceptualizes "diversity" in its social media outreach and how this implementation aligns with the program's stated goals. Specifically, we perform a mixed methods analysis to descriptively capture (1) which underrepresented populations are targeted by the AoURP's social media and (2) how solidaristic messaging is used, if at all, in these calls for participation.AoURP social media posts (n=380) from a 6-month period in 2020-2021 were coded to identify visual depictions and explicit mentions of any "underrepresented in biomedical research" (UBR) categories officially targeted by the program. To then characterize UBR-specific appeals, we performed a thematic analysis of UBR-targeted posts, using a coding scheme that identified unsolidaristic language (ie, appeals to individual benefits) and solidaristic language (ie, appeals to benefitting others, attaining shared goals, and addressing injustices).Among the 10 UBR categories officially recognized by the AoURP, "Race and Ethnicity" (187/380, 49% of posts) and "Age" (71/380, 19%) were the most frequently emphasized, while each of the other remaining categories was rarely invoked (<4/380, 1%). The thematic analysis further identified calls to participate that spanned receiving genetic results (ie, individual benefits), uncovering family and community disease predispositions (ie, benefitting others), improving the future of health (ie, achieving shared goals), and addressing data and health disparities (ie, resolving injustices).In addition to highlighting UBR categories that are more and less emphasized in the AoURP's social media outreach, we also find that the program's messaging indeed resembles a solidaristic appeal to participate. Drawing upon the existing literature on solidarity, we leverage conceptualizations of solidarity as a shared practice grounded in mutuality and bidirectionality to question the AoURP's appeals when institutions fail to fully reciprocate this solidarity. Specifically, we raise concerns about (1) unclear links between participation and addressing health disparities, (2) incomplete acknowledgment of institutions' role in data disparities, and (3) the use of empowerment rhetoric that diverts the onus for correcting these disparities onto participants. Finally, we consider the implications of these issues for future outreach efforts.

  View details for DOI 10.2196/63793

  View details for PubMedID 40844317

• Glass Half Full or Empty: Illuminating the Human Transcriptome - Inspiring Underserved High School Students with an Experiential Biology Laboratory Research Experience Nelson, T., Zhang, D., Sorid, S., Chang, T., Bajaj, N., Viola, J., Heckelman, L., Pollack, R. ELSEVIER. 2024: S787-S788

  View details for DOI 10.1016/j.jbc.2024.107113

  View details for Web of Science ID 001326666903113

• Engineering CRISPR-Associated Transposons for Facile Tagging of Endogenous Proteins Zhang, D., Klompe, S., Walker, M., Lampe, G., Liang, A., Sternberg, S. ELSEVIER. 2024: S481

  View details for DOI 10.1016/j.jbc.2024.106613

  View details for Web of Science ID 001326666902019

• Sinophobic Epidemics in America: Historical Discontinuity in Disease-related Yellow Peril Imaginaries of the Past and Present JOURNAL OF MEDICAL HUMANITIES Zhang, D. 2021; 42 (1): 63-80

  Abstract

  Modern scholarship has drawn hasty and numerous parallels between the Yellow Peril discourses of the 19th- and 20th-century plagues and the recent racialization of infectious disease in the 21st-century. While highlighting these similarities is politically useful against Sinophobic epidemic narratives, Michel Foucault argues that truly understanding the past's continuity in the present requires a more rigorous genealogical approach. Employing this premise in a comparative analysis, this work demonstrates a critical discontinuity in the epidemic imaginary that framed the Chinese as pathogenic. Consequently, those seeking to prevent future disease racialization must understand modern Sinophobia as fundamentally distinct from that of the past.

  View details for DOI 10.1007/s10912-020-09675-x

  View details for Web of Science ID 000620470800002

  View details for PubMedID 33616830

  View details for PubMedCentralID PMC7897726