Lacramioara Bintu

All Publications

• Nanobody-mediated control of gene expression and epigenetic memory. Nature communications Van, M. V., Fujimori, T. n., Bintu, L. n. 2021; 12 (1): 537

  Abstract

  Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we develop a strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene. We show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against HP1 and DNMT1 can silence a reporter gene. Moreover, combining nanobodies together or with other regulators, such as DNMT3A or KRAB, can enhance silencing speed and epigenetic memory. Finally, we use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using nanobodies against chromatin regulators for controlling gene expression and epigenetic memory.

  View details for DOI 10.1038/s41467-020-20757-1

  View details for PubMedID 33483487

• High-Throughput Discovery and Characterization of Human Transcriptional Effectors. Cell Tycko, J. n., DelRosso, N. n., Hess, G. T., Aradhana, n. n., Banerjee, A. n., Mukund, A. n., Van, M. V., Ego, B. K., Yao, D. n., Spees, K. n., Suzuki, P. n., Marinov, G. K., Kundaje, A. n., Bassik, M. C., Bintu, L. n. 2020

  Abstract

  Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit, a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover that Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several domains of unknown function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as ten amino acids. Finally, we report new activator domains, including a divergent KRAB. These results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.

  View details for DOI 10.1016/j.cell.2020.11.024

  View details for PubMedID 33326746

• Mapping chromatin modifications at the single cell level. Development (Cambridge, England) Ludwig, C. H., Bintu, L. n. 2019; 146 (12)

  Abstract

  Understanding chromatin regulation holds enormous promise for controlling gene regulation, predicting cellular identity, and developing diagnostics and cellular therapies. However, the dynamic nature of chromatin, together with cell-to-cell heterogeneity in its structure, limits our ability to extract its governing principles. Single cell mapping of chromatin modifications, in conjunction with expression measurements, could help overcome these limitations. Here, we review recent advances in single cell-based measurements of chromatin modifications, including optimization to reduce DNA loss, improved DNA sequencing, barcoding, and antibody engineering. We also highlight several applications of these techniques that have provided insights into cell-type classification, mapping modification co-occurrence and heterogeneity, and monitoring chromatin dynamics.

  View details for DOI 10.1242/dev.170217

  View details for PubMedID 31249006

• Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements. Nature communications Tycko, J. n., Wainberg, M. n., Marinov, G. K., Ursu, O. n., Hess, G. T., Ego, B. K., Aradhana, n. n., Li, A. n., Truong, A. n., Trevino, A. E., Spees, K. n., Yao, D. n., Kaplow, I. M., Greenside, P. G., Morgens, D. W., Phanstiel, D. H., Snyder, M. P., Bintu, L. n., Greenleaf, W. J., Kundaje, A. n., Bassik, M. C. 2019; 10 (1): 4063

  Abstract

  Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.

  View details for DOI 10.1038/s41467-019-11955-7

  View details for PubMedID 31492858

• Advancing towards a global mammalian gene regulation model through single-cell analysis and synthetic biology Current Opinion in Biomedical Engineering Tycko, J., Van, M. V., Elowitz, M. B., Bintu, L. 2017; 4: 174-193

  View details for DOI 10.1016/j.cobme.2017.10.011