Browsing by Author "Lu, Weiqi"

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    An associative memory Hamiltonian model for DNA and nucleosomes
    (PLOS, 2023) Lu, Weiqi; Onuchic, José N.; Pierro, Michele Di; Center for Theoretical Biological Physics
    A model for DNA and nucleosomes is introduced with the goal of studying chromosomes from a single base level all the way to higher-order chromatin structures. This model, dubbed the Widely Editable Chromatin Model (WEChroM), reproduces the complex mechanics of the double helix including its bending persistence length and twisting persistence length, and the temperature dependence of the former. The WEChroM Hamiltonian is composed of chain connectivity, steric interactions, and associative memory terms representing all remaining interactions leading to the structure, dynamics, and mechanical characteristics of the B-DNA. Several applications of this model are discussed to demonstrate its applicability. WEChroM is used to investigate the behavior of circular DNA in the presence of positive and negative supercoiling. We show that it recapitulates the formation of plectonemes and of structural defects that relax mechanical stress. The model spontaneously manifests an asymmetric behavior with respect to positive or negative supercoiling, similar to what was previously observed in experiments. Additionally, we show that the associative memory Hamiltonian is also capable of reproducing the free energy of partial DNA unwrapping from nucleosomes. WEChroM is designed to emulate the continuously variable mechanical properties of the 10nm fiber and, by virtue of its simplicity, is ready to be scaled up to molecular systems large enough to investigate the structural ensembles of genes. WEChroM is implemented in the OpenMM simulation toolkits and is freely available for public use.
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    Associative Memory Hamiltonian Modeling on DNA, Nucleosomes, and Chromatin
    (2023-09-21) Lu, Weiqi; Onuchic, Jose
    Chromatin, the complex of DNA and proteins, plays a pivotal role in the regulation of gene expression and other cellular processes. Its dynamic organization and conformational plasticity are fundamental to the proper functioning of the genome. Understanding the three-dimensional structure and dynamics of chromatin at various scales is crucial for elucidating the molecular mechanisms underlying gene expression and other biological processes. However, the inherent complexity and multiscale nature of chromatin pose significant challenges for experimental and computational studies. In this thesis, we present the development and application of an Associative Memory Hamiltonian model, the Widely Editable Chromain Model (WEChroM) to gain insights into the structure and dynamics of chromatin fibers and nucleosomes. We begin by introducing the Associative Memory Hamiltonian approach, which leverages prior knowledge of experimentally determined structures to guide molecular dynamics simulations toward biologically relevant conformations. We detail the development of WEChroM for DNA and nucleosomes and demonstrate the model’s capability to capture conformational preferences and mechanical and thermodynamically properties. We investigate the bending and twisting persistence lengths, supercoiling behavior of DNA minicircles, and DNA-protein interactions within the context of nucleosomes. We further discuss the implementation of WEChroM on the OpenMM platform and provide a tutorial on the software. We then apply the WEChroM approach to investigate higher-order chromatin structures. We elucidate organization patterns in nucleosome arrays, explore the 30-nm fiber models, and assess the impact of uniform and non-uniform linker lengths.We discuss the functional implications of nucleosome array organization and compare our theoretical predictions with experimental data. Lastly, we discuss the significance, limitations, challenges, and future directions of the WEChroM approach. In summary, this thesis contributes to the field of computational genomics by providing insights into chromatin structure and dynamics through the development and application of the WEChroM model. Our findings have broad implications for understanding genome function, gene regulation, and the molecular mechanisms behind the phenomenon.
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    A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing
    (Elsevier, 2019) Li, Ang; Lee, Ciaran M.; Hurley, Ayrea E.; Jarrett, Kelsey E.; De Giorgi, Marco; Lu, Weiqi; Balderrama, Karol S.; Doerfler, Alexandria M.; Deshmukh, Harshavardhan; Ray, Anirban; Bao, Gang; Lagor, William R.; Bioengineering
    Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans.