Nourreddine S, Doctor Y, Dailamy A
… +19 more, Lee YH, Hansen JN, Chinn R, Forget A, Polacco B, Muralidharan M, Sigaeva A, Sunder S, Pan E, Gao J, Chen JY, Clark T, Parker J, Obernier K, Metallo C, Ideker T, Lundberg E, Krogan N, Mali P
Comprehensively mapping the relationship between genotype and phenotype offers essential insights into how a cell's state arises from its genetic components. Toward this goal, we generated an expressed genome-scale CRISP...Comprehensively mapping the relationship between genotype and phenotype offers essential insights into how a cell's state arises from its genetic components. Toward this goal, we generated an expressed genome-scale CRISPRi perturbation cell atlas in KOLF2.1J human induced pluripotent stem cells, mapping transcriptional phenotypes associated with 11,692 perturbed genes across >2.5 million single cells. Using correlations among perturbed phenotypes, we created a cell map of the pluripotent state, demonstrating rich recapitulation of functionally related protein complexes. We then explored the atlas to uncover metabolic factor ZBTB41 and pluripotency regulator RNF7, validating their functions through metabolic tracing, immunofluorescence and protein-protein interaction assays. Lastly, we leveraged the atlas to generate a genome-scale screen of A-to-I RNA-editing modulators assayed through direct transcriptome-wide RNA editing, uncovering and mechanistically validating DBR1 as a potent regulator. Taken together, our data provide a comprehensive resource for interrogating the regulatory networks governing pluripotency, which is accessible at https://y-doctor.github.io/KOLF2.1J_Perturbation_Cell_Atlas/ .
Prime editing has not been established in filamentous fungi, which are major ecological contributors and industrial hosts with vast biosynthetic capacity. Here we develop fPE7max, a prime editing platform optimized for f...Prime editing has not been established in filamentous fungi, which are major ecological contributors and industrial hosts with vast biosynthetic capacity. Here we develop fPE7max, a prime editing platform optimized for fungi, which supports different edit types, including base substitutions and defined small insertions or deletions, with an average editing efficiency approaching 90%, across diverse genomic loci and species. fPE7max further enables larger insertions of up to 1 kb and deletions of up to 10 kb. We perturb upstream open reading frames in the pleiotropic regulator gene, laeA, to modulate metabolic output across multiple fungal species. Metabolomic profiling reveals activation of previously lowly biosynthetic pathways, leading to the identification of 18 metabolites, including 8, to our knowledge, previously unreported structures, 3 of which with cytotoxic activity. These results establish fPE7max as an efficient platform for genome engineering in filamentous fungi and show upstream open reading frame editing as a strategy for modulating endogenous regulatory networks and accessing the fungal chemical repertoire.
Fauser F, Arangundy-Franklin S, Davis JE
… +27 more, Liu L, Schmidt NJ, Rodriguez L, Xia DF, Nguyen N, Zhou Y, Scarlott NA, Truong LN, Mureli R, Tan IS, Sajuthi S, Hinkley SJ, Kadam BN, Lam S, Bourgeois B, Tait E, Qasim M, Vaidya V, Chen A, Nguyen A, Bendaña YR, Shivak DA, Li P, Reik A, Paschon DE, Davis GD, Miller JC
Serine integrases can precisely integrate large DNA constructs into desired chromosomal sites but only if their natural target site is first installed into the recipient genome. Here, to retarget serine integrases to a d...Serine integrases can precisely integrate large DNA constructs into desired chromosomal sites but only if their natural target site is first installed into the recipient genome. Here, to retarget serine integrases to a desired genomic site, we develop a modular integrase (MINT) system for genome editing. Through a combination of structural modeling, single-round directed evolution and screening in human cells, we retargeted the specificity of the serine integrase Bxb1. We demonstrate the therapeutic potential of the MINT system by retargeting Bxb1 to the human AAVS1 and TRAC loci, where wild-type Bxb1 has no detectable activity. By combining MINT constructs with both known activity-increasing Bxb1 mutants and zinc-finger DNA-binding domains, we achieve efficiencies of 29% at the AAVS1 locus and 35% at the TRAC locus in K562 cells. To further demonstrate clinical potential, we achieved 29% GFP integration efficiencies at the TRAC locus in human T cells.
AlphaFold3 predicts highly accurate protein structures from sequence but tends to collapse to a single dominant conformation, even when the underlying structure is inherently heterogeneous. Moreover, its predictions are...AlphaFold3 predicts highly accurate protein structures from sequence but tends to collapse to a single dominant conformation, even when the underlying structure is inherently heterogeneous. Moreover, its predictions are oblivious to experimental conditions that can alter local sequence conformation. In this work, we show that AlphaFold3 can be guided to match data obtained by nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and cryogenic electron microscopy (cryo-EM) experiments and combinations thereof. Our approach can also incorporate data that explicitly report on dynamics, such as site-resolved order parameters. We demonstrate that this methodology generates compact structural ensembles whose ensemble-averaged observables agree with experiment, with fewer distance restraint violations than traditionally resolved NMR structures and with unmodeled alternate conformations uncovered in electron density. This methodology paves the way for experimentally aware predictive models that generate structural ensembles consistent with the measurements, potentially over multiple modalities, and that can be further refined toward thermodynamically grounded ensembles by incorporating energetics.
Extracellular vesicles (EVs) have emerged as promising biomarkers for monitoring physiological homeostasis and pathological progression. However, current analytic methods face limitations in preserving spatial informatio...Extracellular vesicles (EVs) have emerged as promising biomarkers for monitoring physiological homeostasis and pathological progression. However, current analytic methods face limitations in preserving spatial information about EVs and their intricate connections to parental and recipient cells. Here, we present Spatial-EV-seq, a method for in situ spatial profiling of EVs within their native microenvironmental context. Spatial-EV-seq uses an antibody-engineered capture interface to preserve EVs' spatial distribution, followed by rolling circle amplification with EV surface-binding aptamers, enabling fluorescence imaging and molecular profiling of individual EVs. The method integrates ultrasensitive EV profiling, molecular subtyping and high-resolution spatial mapping with transcriptomics to resolve location-specific EV-cell communication networks. In an anti-PD1-treated breast cancer mouse model, we uncover a spatially orchestrated immunosuppressive axis: PDL1 EV-enriched zones drive CD8 T cell dysfunction, establishing immune-privileged niches, whereas PDL1 EV-depleted regions preserve immunocompetence and therapeutic sensitivity. Spatial-EV-seq offers insights into EV-mediated mechanisms and unlocks avenues for precision diagnostics and therapeutics.
Di Carlo D, Morsut L, McCain ML
… +13 more, Wright HJ, Abedi M, Yamada-Hunter SA, Zhang JZ, Backus K, Chung E, Wang Y, Rando TA, Cai L, Thomson M, Elowitz MB, Lee JK, Witte O
Efforts to systematically understand how cell interactions tune tissue-level function have motivated transformative advances in single-cell transcriptomics and spatial profiling. Although these technologies can measure m...Efforts to systematically understand how cell interactions tune tissue-level function have motivated transformative advances in single-cell transcriptomics and spatial profiling. Although these technologies can measure molecular states in individual cells and their spatial mapping within tissues, they also reveal that there exists a fundamental knowledge gap of how cells influence each other in context. In this Perspective, we propose an initiative to map and engineer the human cell-cell interactome: a functional atlas of how all major human cell types communicate. We highlight how recent innovations can make this vision achievable. As a first moonshot, we propose the 'Billion Cell×Cell Project', which systematically characterizes the outcomes of defined cell-cell dyads across diverse cell types and conditions. We envision this multistage initiative will produce progressively deeper insights and unlock additional avenues for therapeutic discovery. We call on the scientific community to join us in building the tools, datasets and models that will decode and rewrite the language of life between cells.
Obtaining antibodies to specific protein targets is a widely important yet experimentally laborious process. Meanwhile, computational methods for antibody design have been limited by low success rates that require resour...Obtaining antibodies to specific protein targets is a widely important yet experimentally laborious process. Meanwhile, computational methods for antibody design have been limited by low success rates that require resource-intensive screening. Here we introduce Germinal, a broadly enabling generative pipeline that designs antibodies against specific epitopes with nanomolar binding affinities while requiring only low-n experimental testing. Our method co-optimizes antibody structure and sequence by integrating a structure predictor with an antibody-specific protein language model to perform de novo design of functional complementarity-determining regions onto a user-specified structural framework. When tested against four diverse protein targets, Germinal designed functional antibodies across all targets and binder formats, testing only 43-101 designs for each antigen. Validated designs also exhibited robust expression in mammalian cells and high sequence and structural novelty. We provide open-source code and full computational and experimental protocols to facilitate wide adoption.
Traditional approaches for DNA insertion into plant genomes using Agrobacterium tumefaciens result in random integration. Newer genetic engineering methods based on nucleases, prime editors, transposases and recombinases...Traditional approaches for DNA insertion into plant genomes using Agrobacterium tumefaciens result in random integration. Newer genetic engineering methods based on nucleases, prime editors, transposases and recombinases extend capabilities but remain constrained with low efficiencies, off-target integration or limited payload size. Here we adapt the avian Taeniopygia guttata R2 protein (R2Tg) for targeted DNA insertion into plant genomes by engineering R2Tg expression cassettes and RNA payloads carrying intron-disrupted reporters, with optimized ribosomal DNA homology arms and untranslated regions. In Arabidopsis thaliana protoplasts, Nicotiana benthamiana leaves and Solanum lycopersicum seedlings, our R2Tg editor system achieves targeted insertion of full-length payloads ranging from 2.2 kb to 5 kb. In Nicotiana benthamiana leaves, integration occurs, on average, at 1 copy per genome, which is 30 times more efficient than that achieved by Cas9 homology-directed repair. This work establishes an R2Tg ribonucleoprotein platform for targeted DNA insertion into plant genomes, using a multicopy genomic safe-harbor site to enable efficient addition of multikilobase genes.
Precise integration of multikilobase DNA fragments remains a major technical barrier in plants. Here we introduce non-long terminal repeat (non-LTR) R2 retrotransposons as a versatile system for targeted gene integration...Precise integration of multikilobase DNA fragments remains a major technical barrier in plants. Here we introduce non-long terminal repeat (non-LTR) R2 retrotransposons as a versatile system for targeted gene integration in plants. We reconstituted R2 activity in Nicotiana benthamiana and benchmarked insertion efficiency and fidelity using a TMV-based episomal reporter system. We demonstrate site-specific integration of GFP (2.2 kb) and recombinase-compatible landing pads (0.6 kb) into 28S rDNA arrays, with intact cassette insertion frequencies up to 75% and 53%, respectively. To temporally constrain donor availability and avoid DNA intermediates, we combined in planta effector expression with recombinant RNA virus-mediated donor delivery. We apply R2 retrotransposons for targeted insertion of resistance cassettes within the rDNA of rice callus, achieving integration efficiencies up to 17%. These results position R2 retrotransposons as a double-strand break-free system for RNA-templated insertion of multikilobase gene cassettes at rDNA loci, for safe-harbor trait stacking in plants with potential applications in crop improvement and synthetic biology.