Angela Pogson

All Publications

• CRISPR-Cas9 screens reveal regulators of ageing in neural stem cells. Nature Ruetz, T. J., Pogson, A. N., Kashiwagi, C. M., Gagnon, S. D., Morton, B., Sun, E. D., Na, J., Yeo, R. W., Leeman, D. S., Morgens, D. W., Tsui, C. K., Li, A., Bassik, M. C., Brunet, A. 2024

  Abstract

  Ageing impairs the ability of neural stem cells (NSCs) to transition from quiescence to proliferation in the adult mammalian brain. Functional decline of NSCs results in the decreased production of new neurons and defective regeneration following injury during ageing1-4. Several genetic interventions have been found to ameliorate old brain function5-8, but systematic functional testing of genes in old NSCs-and more generally in old cells-has not been done. Here we develop in vitro and in vivo high-throughput CRISPR-Cas9 screening platforms to systematically uncover gene knockouts that boost NSC activation in old mice. Our genome-wide screens in primary cultures of young and old NSCs uncovered more than 300 gene knockouts that specifically restore the activation of old NSCs. The top gene knockouts are involved in cilium organization and glucose import. We also establish a scalable CRISPR-Cas9 screening platform in vivo, which identified 24 gene knockouts that boost NSC activation and the production of new neurons in old brains. Notably, the knockout of Slc2a4, which encodes the GLUT4 glucose transporter, is a top intervention that improves the function of old NSCs. Glucose uptake increases in NSCs during ageing, and transient glucose starvation restores the ability of old NSCs to activate. Thus, an increase in glucose uptake may contribute to the decline in NSC activation with age. Our work provides scalable platforms to systematically identify genetic interventions that boost the function of old NSCs, including in vivo, with important implications for countering regenerative decline during ageing.

  View details for DOI 10.1038/s41586-024-07972-2

  View details for PubMedID 39358505

• DNA-guided transcription factor cooperativity shapes face and limb mesenchyme. Cell Kim, S., Morgunova, E., Naqvi, S., Goovaerts, S., Bader, M., Koska, M., Popov, A., Luong, C., Pogson, A., Swigut, T., Claes, P., Taipale, J., Wysocka, J. 2024

  Abstract

  Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution.

  View details for DOI 10.1016/j.cell.2023.12.032

  View details for PubMedID 38262408

• DNA-guided transcription factor cooperativity shapes face and limb mesenchyme. bioRxiv : the preprint server for biology Kim, S., Morgunova, E., Naqvi, S., Bader, M., Koska, M., Popov, A., Luong, C., Pogson, A., Claes, P., Taipale, J., Wysocka, J. 2023

  Abstract

  Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how 'Coordinator', a long DNA motif comprised of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, while HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in shared regulation of genes involved in cell-type and positional identities, and ultimately shapes facial morphology and evolution.

  View details for DOI 10.1101/2023.05.29.541540

  View details for PubMedID 37398193

  View details for PubMedCentralID PMC10312427

• Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors. eLife Replogle, J. M., Bonnar, J. L., Pogson, A. N., Liem, C. R., Maier, N. K., Ding, Y., Russell, B. J., Wang, X., Leng, K., Guna, A., Norman, T. M., Pak, R. A., Ramos, D. M., Ward, M. E., Gilbert, L. A., Kampmann, M., Weissman, J. S., Jost, M. 2022; 11

  Abstract

  CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal non-specific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.

  View details for DOI 10.7554/eLife.81856

  View details for PubMedID 36576240

• Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq CELL Replogle, J. M., Saunders, R. A., Pogson, A. N., Hussmann, J. A., Lenail, A., Guna, A., Mascibroda, L., Wagner, E. J., Adelman, K., Lithwick-Yanai, G., Iremadze, N., Oberstrass, F., Lipson, D., Bonnar, J. L., Jost, M., Norman, T. M., Weissman, J. S. 2022; 185 (14): 2559-+

  Abstract

  A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes to predict the function of poorly characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena-from RNA processing to differentiation. We leverage this ability to systematically identify genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene and cellular function.

  View details for DOI 10.1016/j.cell.2022.05.013

  View details for Web of Science ID 000961505000001

  View details for PubMedID 35688146

  View details for PubMedCentralID PMC9380471

• Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell Nunez, J. K., Chen, J., Pommier, G. C., Cogan, J. Z., Replogle, J. M., Adriaens, C., Ramadoss, G. N., Shi, Q., Hung, K. L., Samelson, A. J., Pogson, A. N., Kim, J. Y., Chung, A., Leonetti, M. D., Chang, H. Y., Kampmann, M., Bernstein, B. E., Hovestadt, V., Gilbert, L. A., Weissman, J. S. 2021

  Abstract

  A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.

  View details for DOI 10.1016/j.cell.2021.03.025

  View details for PubMedID 33838111