Research Overview

We develop biotechnologies for genome engineering and synthetic biology to flexibly program and manipulate the genomic codes of life. To expand our bioengineering toolbox and to understand the function of the genome in development and disease, we developed a suite of CRISPR tools, including the nuclease-deactivated dCas molecules from the natural CRISPR-Cas system for transcription activation or repression (CRISPRi/a), live-cell genome imaging (LiveFISH), and manipulating the spatial genome organization (CRISPR-GO). We appreciate the vast macromolecules (DNA, RNA, and proteins) that have been evolved throughout billions of years of history and aim to harness these naturally occurring molecules as tools. We appreciate the power of directed evolution to obtain novel molecules that have been discovered or evolved in nature. Topics that are particularly interesting to us:

New tools for engineering human genome, broadly defined Example: dCas9; CRISPRi; CRISPRa; LiveFISH; CRISPR-GO; CasMINI

The ideal genomic tools are those that can control any gene in any cell type for any mode of regulation (transcription, translation, epigenetics, spatial, temporal, multiplexed, quantitative, inducible, and conditional). Examples include:

· dCas9: Development of the first nuclease-dead dCas9 molecule for gene expression regulation (Qi Cell 2013)

· CRISPRi and CRISPRa: Development of CRISPRi and CRISPRa in eukeryotic cells, including mammalian and human cells (Gilbert Cell 2013)

· Multiplexed CRISPRi/a: Development of CRISPR-mediated systems by recruiting RNA aptamer-engineered guide RNAs for multiplexed gene regulation (Zalatan Cell 2015; Gao Nat Method 2016)

· GPCR-modulated genome editing: Coupling CRISPR to signal detection of broad antigens and ligands by GPCR or promoters (Kipniss Nat Comm 2017; Kempton Mol Cell 2020)

· Live-cell imaging and LiveFISH: imaging of genome dynamics in cells (Chen Cell 2013; Wang Science 2019)

· CRISPR-GO: manipulating the 3D genome structure within the nucleus (Wang Cell 2018)

· CasMINI: Miniaturized CRISPR molecule for genome and epigenome editing (Xu Mol Cell 2021). For guide RNA design, go to here: https://www.casmini-tool.com/

· HyperCas12a: An engineered hyper-efficient Cas12a for multiplexed gene modulation or editing in vivo (e.g., mouse) (Guo Nature Cell Biology 2022)

Synthetic mammalian biology: designing DNA and molecules to obtain desired cellular functions Example: Boolean circuits; Anti-CRISPR circuits; ChaCha receptors; better CAR T cell design

The genome controls its function via multiplex routes ranging from small to large physical scales: DNA sequence, epigenetic modifications, cis-regulatory elements, DNA looping, TADs (topologically associated domains), and nuclear domains, together control the genome in a coordinated and dynamic manner. Modern genomics research aims to develop a coherent understanding spanning these scales to provide a complete picture on how the genome controls its function on multiple levels. We aim to apply the new tools to understand how to 3D genome structure affects function. We are applying diverse genome engineering tools, based on high-throughput perturbation and measurement, to study how the spatial and temporal organization of the genome affects gene expression, epigenetics chemical modifications, and cellular functions. In the past, we demonstrated that the high-throughput control of the genome is a powerful approach. Using pairwise CRISPRi-based repression, we identified a gene network that causes synthetic lethality in cancer cells (Du Nature Methods 2017). Using single- and double-CRISPRa screens, we identified factor combinations that can efficiently reprogram fibroblast to neuron (Y Liu Cell Stem Cell 2018). We believe the new genome engineering tools will unravel new information about key factors that control the genome function.

Gene and cell therapy: novel approaches for using genes for treating complex diseases Example: regenerative medicine; aging

We combine genome engineering with synthetic biology to design the mammalian genome for therapeutics. For example, we design and engineer cells to sense multiple antigen inputs and carry out therapeutic functions (Kipniss Nat Comm 2017; Kempton Mol Cell 2020). We engineered molecular devices (ChaCha) that couple human GPCR receptor function to dCas9 effectors, such that cells can detect extracellular signals and control gene expression. We show CRISPRa-mediated activation of targeted genome and epigenome sites can reprogram fibroblast to induced pluripotent stem cells (P Liu Cell Stem Cell 2018), or fibroblast to neuron (Y Liu Cell Stem Cell 2018). We use genome engineering to explore its function as genetic sensors or wires that allow connecting synthetic circuits to therapeutic functions.

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Research Overview