The ON-OFF switch of genes is critical for cell fate decision, development and human disease. Our group will combine single cell genomics and cutting-edge microscopy technology to identify what genes are turned on or off in cell type-specific or disease-specific manners. How are these genes precisely controlled in space and time? We will use CRISPR-based live cell DNA and RNA tracing system to understand the spatiotemporal regulation of genes during the physiological process, and we will harness CRISPR-based editing technology to reverse aberrant gene expression in the disease models.

Genome Imaging

The CRISPR-Cas9 system has been repurposed for genome imaging (Chen et al., Cell 2013). We have developed a series of genome imaging tools, such as multicolor CRISPR (Ma et al., PNAS 2015), CRISPR-Broccoli (Ma et al., J Cell Biol 2016), CRISPRainbow (Ma et al., Nat Biotechnol 2016), CRISPR-Sirius (Ma et al., Nat Methods 2018).

Gene expression is precisely controlled in space and time during cell proliferation and differentiation. It is still unclear how regulatory genes are relocated from repressive domains to active domains in response to cellular stimuli, or vice versa. CRISPR-based live-cell DNA and RNA imaging are perfect tools to study the spatiotemporal regulation of genes during the physiological process or in the pathological context. We are developing robust CRISPR-based imaging systems to label individual regulatory elements or genes. These approaches will allow us to understand the interplay between chromatin dynamics and transcriptional regulation.

Genome Editing

The bacterial CRISPR-Cas9 system has been repurposed for genome engineering in eukaryotic cells. How does the CRISPR-Cas9 system find specific target in the genome not well defined in living cells. It has been shown that the CRISPR-Cas9 genome interrogation is subjected to 3D diffusion in living cells (Knight et al., Science 2015), and the searching process is slow but in a Cas9/guide RNA dose-dependent manner (Jones et al., Science 2017). We have found that CRISPR guide RNA is extremely unstable in the absence of Cas9. Meanwhile CRISPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time (Ma et al., JCB 2016)

Prime editing developed by David Liu’s lab is a very promising tool for base substitution, small fragment insertion and deletion with high precision (Anzalone et al., Nature 2019). We have found the pegRNAs are extremely unstable even in the present of Cas9 during prime editing. We are applying for the above principles to stabilize pegRNA, optimize Cas9/pegRNA assembly, improve DNA interrogation and prime editing. 

Epigenetic Regulation

Epigenetic regulation is important for cell fate decision, development and human disease. We have developed an EpiGo (Epigenetic perturbation induced Genome organization) system, which allows us simultaneously introduce epigenetic modifications and track genome organization. EpiGo-KRAB-induced heterochromatin-like domain does not result in widespread gene repression except a small set of genes with concurrent loss of H3K4me3 and H3K27ac (Feng et al., Genome Biol 2020).

Other epigenetic modifications such as H3K27me3, H3K4me3, m5C and H2Aub etc are also important for development and disease. We are systematically investigating the roles of ectopically epigenetic modifications on transcriptional activity and genome organization by the EpiGo system in living cells.

Phase Separation

The cell nucleus is a crowding microenvironment full of nuclear nanobodies (David Spector, Cell 2006). The Nanobodies mainly consist of nuclear proteins, regulatory and nascent RNAs, and/or chromatin DNA. It has been shown that many of these nanobodies, named condensates, are also involved in transcriptional regulation by liquid-liquid phase separation (LLPS) (Strom et al., Nature 2017; Sabari et al., Science 2018; Cho et al., Science 2018). However it is still unclear what is the composition of transcriptionally active or repressive nanobodies. Particularly the role of RNA or chromatin DNA in condensate formation is not well defined. The function of condensates in transcriptional regulation is far from a full understanding.

We use super-resolution microscopy and single molecule tracking of protein, RNA and chromatin DNA simultaneously in living cells. Our long-term goal is to understand how nuclear condensates are dynamically regulated and their contributions to transcriptional regulation in space and time.