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Abstract The spatial organization and dynamics of a genome are central to gene regulation. While a comprehensive understanding of chromatin organization in the human nucleus has been achieved using fixed-cell methods, measuring the dynamics of specific genomic regions over extended periods in individual living cells remains challenging. Here, we present a robust and fully genetically encoded system for fluorescent labeling and long-term tracking of any accessible non-repetitive genomic locus in live human cells using fluorogenic and replenishable nanobody array fusions of theStaphylococcus aureusdCas9, and compact polycistronic single guide (sg)RNAs. First, we characterize the selectivity and photostability of our probes, enabling genome-wide visualization of chromatin dynamics at locally repetitive elements. Next, through multiplexed expression of 8–10 sgRNAs from polycistronic cassettes, we demonstrate efficient and sustained labeling of non-repetitive loci, enabling high-fidelity tracking of gene-proximal regions at exceptional spatial and temporal resolution. Finally, by correlating chromatin mobility with transcriptional activity at multiple genes, we find that local chromatin dynamics at 20 Hz are gene-specific and not necessarily dependent on transcription. Our approach is versatile, minimally invasive, and scalable, enabling multiplexed imaging of regulatory element dynamics involved in gene control, with broad applicability across diverse biological systems and disease contexts. 

Over the past two decades Biomedical Engineering has emerged as a major discipline that bridges societal needs of human health care with the development of novel technologies. Every medical institution is now equipped at varying degrees of sophistication with the ability to monitor human health in both non-invasive and invasive modes. The multiple scales at which human physiology can be interrogated provide a profound perspective on health and disease. We are at the nexus of creating “avatars” (herein defined as an extension of “digital twins”) of human patho/physiology to serve as paradigms for interrogation and potential intervention. Motivated by the emergence of these new capabilities, the IEEE Engineering in Medicine and Biology Society, the Departments of Biomedical Engineering at Johns Hopkins University and Bioengineering at University of California at San Diego sponsored an interdisciplinary workshop to define the grand challenges that face biomedical engineering and the mechanisms to address these challenges. The Workshop identified five grand challenges with cross-cutting themes and provided a roadmap for new technologies, identified new training needs, and defined the types of interdisciplinary teams needed for addressing these challenges. The themes presented in this paper include: 1) accumedicine through creation of avatars of cells, tissues, organs and whole human; 2) development of smart and responsive devices for human function augmentation; 3) exocortical technologies to understand brain function and treat neuropathologies; 4) the development of approaches to harness the human immune system for health and wellness; and 5) new strategies to engineer genomes and cells. 

Cytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted C , existing base editors fail to discriminate them and edit both Cs. To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5′-C C -3′ motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity.