Past research

Gene regulation operates at multiple levels: from the three-dimensional genome perspective, the folding of the genome in three-dimensional space allows interactions between different functional non-coding and coding regions. For example, enhancers can be brought near promoters, thereby activating the expression of specific genes. From the viewpoint of epigenetic modifications, the chemical modifications of DNA or histones play a crucial regulatory role in gene expression. Similarly, post-transcriptional processing of RNA is also an extremely important regulatory process. For instance, through RNA alternative splicing, transcripts with different exons can be generated, leading to proteins with different amino acid sequences and functions. These intricate gene regulation processes are interconnected and in a constant state of dynamic change, allowing a limited number of genes to produce a myriad of possibilities. However, when regulation goes awry, gene function may not be correctly expressed, potentially causing physiological disruptions or even diseases.

Professor Cheng has long been dedicated to studying the mechanisms of epigenetics, three-dimensional genome folding, and RNA splicing in cellular reprogramming, differentiation, and cancers. In one study, Cheng analyzed the epigenetic marks of enhancers and found that enhancers can exist in different epigenetic states. The transition of states regulates gene expression during stem cell differentiation and predicts the fate of downstream cells in differentiation. This research established H3K27ac modification as a marker for active enhancers. In two other studies, Cheng analyzed alternative splicing of RNA during breast cancer metastasis and erythropoiesis, identifying prognostic splicing markers as well as splicing factors that regulate these two important processes.

As our understanding of gene regulatory mechanisms grows, there is an increasing demand for tools for editing and rewriting the genome or transcriptome to further establish cause-effect relationships between genetic elements. In 2012, the efficient genome editing tool CRISPR technology emerged. Based on CRISPR technology, Cheng developed various editing tools for the genome and epigenome. In 2013, the CRISPR-on system for RNA-induced gene activation/epigenetic editing was published. To further enhance the flexibility and efficiency of CRISPR for epigenetic editing, Cheng created the Casilio system, which supports simultaneous editing of multiple sites for different epigenetic modifications. Using the Casilio system, the research group developed Casilio-ME, an efficient DNA methylation editing tool, and Casilio-Imaging, a live-cell chromatin imaging tool. With Casilio-Imaging, the group successfully tracked the dynamic interaction between enhancers and promoters and, through collaboration, conducted real-time tracking of extrachromosomal DNA (ecDNA) in cancer cells, revealing uneven mitotic distribution and hub formation behaviors of ecDNA. For RNA-level editing tools, the research group established the CASFx system based on CRISPR-Cas13 for RNA splicing editing and the CREST editing system that supports simultaneous editing of multiple RNAs with different effects. In another collaboration, the group used multi-omics analysis to elucidate the molecular mechanisms of fusion proteins affecting the three-dimensional genome and gene expression in supratentorial ependymoma.

Future research directions

The research group primarily focuses on scientific questions related to cell fate transitions during development and disease processes, as well as the gene regulatory mechanisms involved in fate determination. This includes epigenetic modifications, three-dimensional genome structure, transcriptional regulation, and RNA processing.

High-throughput sequencing technologies in the last two decades have provided extensive and detailed data for various gene regulatory aspects. However, most of the data obtained from these different gene regulatory levels can only establish correlations between molecular events and phenotypes, making it challenging to precisely elucidate causation or identify decisive events within the complex gene networks.

To overcome these challenges, tools that can "write" and accurately modify molecular states in live cells or animals are needed. These tools must be capable of simultaneously modifying multiple molecules in different ways and scalable to high-throughput experiments to support comprehensive exploration of the entire genome. The first direction of the research group is to develop "writing" technologies for the genome, epigenome, and transcriptome that meet these requirements. These technologies are combined with high-throughput and single-cell sequencing techniques to study the gene regulatory mechanisms during development and disease processes.

On the other hand, most "reading" technologies to date, i.e., sequencing methods, are designed for population of fixed cells, providing only snapshots of their states. However, many biological processes are heterogeneous, dynamic, or possess memory (requiring continuous observation of the same cells). The second direction of the research group is to develop novel real-time “reading" technologies to track the dynamic changes of individual live cells, focusing on the dynamic relationships between three-dimensional genome structure, epigenetic marks, and gene expression heterogeneity, stochasticity, memory, and the diverse phenotypic outcomes. By combining real-time “reading" and "writing" components along with molecular logic gates, the group aims to construct smart gene therapies that can simultaneously sense multiple cell states and in turns elicit specific therapeutic effects.

Direction 1: Development and application of novel "writing" technologies to investigate gene regulatory networks.

Project 1.1: Develop a comprehensive, multitasking, and multifunctional epigenetic and gene editing toolbox.

Sequencing technology elucidates comprehensive molecular changes in normal and disease states. Using high-throughput sequencing technology to "read" molecular events at different gene regulatory levels allows us to depict complex gene regulatory networks. To validate these observations and identify key players, researchers need the ability to highly precisely modify (i.e., "write") the genome and epigenome.

The CRISPR/Cas system was initially applied to edit genomic sequences (genome editing technology). Modified versions of CRISPR/Cas with Cas9 and gRNA can regulate gene expression states or modify epigenetic marks, known as epigenetic editing technology. Epigenetic editing with CRISPR broadens the possibilities for basic research and translational applications. However, earlier-published epigenetic editing tools cannot multiplex or multitask to edit complex gene networks. The objective of this project is to develop a comprehensive epigenome editing toolbox based on the Casilio platform. The Casilio system combines CRISPR/dCas9 with the Pumilio (PUF) RNA-binding protein effector. In the Casilio system, gRNA can recruit different effectors to the target using specific RNA scaffold sequences, achieving multitasking effector dominance. Additionally, PUF's flexibility provides significant expandability for the Casilio system. Specific goals include: (1) Developing a comprehensive epigenetic editing toolbox. Casilio can use these editing modules for multiplex and combinatorial editing, such as editing different epigenetic marks at different genomic loci simultaneously, and editing multiple marks at each locus. (2) Constructing cell lines and animal models containing Casilio functionality. Casilio and common modules (e.g., genome editing, gene activation/inhibition, DNA methylation/demethylation) will be "installed" into commonly used cell lines and animal models (such as mice). Researchers only need to introduce gRNAs to achieve epigenetic editing. (3) Building a whole-genome gRNA library for gene regulatory elements (such as enhancers and insulators), supporting reverse epigenetic screening experiments.

FIG 1. (1) Build molecular tools for editing various epigenetic marks. (2) Construct cell lines and animal models with editing capabilities. (3) Create a genome-wide regulatory element gRNA library for high-throughput functional screening.

Project 1.2: Establish a CRISPR-Based Artificial RNA Editing and Regulation Platform

This project extends CRISPR editing tools to the RNA level. The aim is to design a series of artificial RNA effectors that control and edit RNA molecules throughout the entire RNA lifecycle, including RNA splicing, end processing, translation, localization, stability, and transcriptome modifications (such as m6A). Specific objectives include: (1) Designing a series of Artificial CRISPR-based RNA Effectors (ACRE) for splicing, end processing, translation, stability, and m6A modification of target RNA molecules. (2) Implementing chemical/light-induced tunable control to enhance flexibility in applications. (3) Developing gRNA libraries for various RNA elements, targeting different types of cis-regulatory elements in known coding and non-coding RNA categories within the transcriptome, including RNA splicing elements, 5' and 3' untranslated regions (UTRs), upstream open reading frames (uORFs), etc.

FIG 2. Build an RNA regulation platform (ACRE) to selectively control the processing and functions of RNA molecules at various levels.

Project 1.3: Integrating Single-Cell Analysis and Epigenetic Editing to Study Cell State Transitions, Disease Mechanisms, and Create Novel Gene Therapies

The research group will use single cell (sc)RNA-seq technology to obtain time-series transcriptomes of individual cells undergoing reprogramming, differentiation, trans-differentiation, and epithelial-mesenchymal transition. Time-series data and epigenetic profiles will be used to construct gene regulatory network models. Casilio or ACRE will then be used to perturb crucial nodes in the network to study the gene regulatory mechanisms underlying cell state transitions. Additionally, the group will apply CRISPR tools to regulate cancer gene networks, study the splicing factor network in breast cancer, and explore epigenetic regulation in other diseases (such as diabetes). The potential use of Casilio gene activation in treatment is also being investigated, such as using Casilio to activate specific genes in cancer cells, leading to in vitro cancer cell death and in vivo cancer regression. In collaboration, the group is analyzing transcriptomes, epigenomes, and three-dimensional genomes induced by fusion proteins in cancer cells. CRISPR whole-genome knockout screening experiments are ongoing to discover genes required for cancer cell proliferation. Through collaboration, the protein interaction network of this fusion protein is being analyzed, and the protein structure of the fusion protein is being resolved. The goal is to combine systems biology approaches with drug screening and repurposing to identify actionable targets and drugs that can inhibit or eliminate cancer cells. The group will use Casilio epigenetic editing modules to modulate epigenetic states of cis-regulatory elements discovered by multi-omic analysis, studying their importance in cancer cell gene expression programs and cellular phenotypes.

Direction 2: Development and Application of Real-time “Reading” Technology to Investigate 3D Genome and Gene Expression Dynamics and Create Smart Therapeutics Combining Reading and Writing Techniques

Project 2.1: Development of Live Cell Imaging Technology to Study Chromatin Interaction and Folding Dynamics

The three-dimensional structure of the genome is heterogeneous and dynamic. This project involves designing fluorescent probes for labeling non-repetitive DNA sequences in living cells. For instance, using different fluorophores to label pairs of enhancers and promoters, the research explores their dynamic interactions in hundreds of single cells through live-cell fluorescence confocal microscopy. Published CRISPR imaging methods effectively label repetitive sequences, but labeling non-repetitive sequences poses a significant challenge due to the low signal-to-noise ratio (SNR) at target sites. Casilio-Imaging, using multiple PUF binding sites on the gRNA scaffold, recruits multiple fluorophores, significantly improving SNR. This enhancement allows a single gRNA to label non-repetitive sequences, greatly increasing the efficiency of chromatin imaging. Multiple probes can be simultaneously deployed to track continuous DNA regions, studying the folding dynamics of DNA regions. To facilitate widespread application, the research group is designing new software for imaging gRNA design and expanding the fluorescent color palette to allow tracking of more sites simultaneously. The group will apply these imaging technologies to address unresolved questions about the three-dimensional genome structure: (1) Dynamics and cell-to-cell heterogeneity of paired and multi-way interactions. Multiple cis-regulatory elements (such as enhancers and promoters) have been identified in sequencing experiments to interact in pairs or multi-directionally. Do they represent stable interactions between all individual cells, or is there a dynamic cycling "merged" snapshot view among multiple possible states, where different interaction combinations may occur at any given time in each individual cell? Observing multiple interacting sites with multi-colored Casilio imaging will clarify these questions; (2) Chromatin loop extrusion. Chromatin loops are formed by architectural factors (such as adhesive proteins) that bind to DNA and coil into DNA to form loops. Several models have been proposed by fellow scientists about the dynamics of loop extrusion processes, namely unidirectional, bidirectional, or mixed models. These models stem from theoretical analyses of sequencing data or imaging of in vitro reconstructed complexes of DNA and architectural factors, thus omitting the dynamic and heterogeneous nature of loop formation in native chromatin under physiological conditions. To understand the process of loop formation in native chromatin under physiological conditions, the research group will use Casilio-Imaging to image chromatin in live cells; (3) Impact of histone modifications or DNA methylation on the dynamics of chromatin interactions. How do histone modifications and DNA methylation affect the frequency and dynamics of chromatin interactions at specific gene loci? By utilizing Casilio tools and simultaneously performing epigenetic editing to alter the methylation/histone modification of enhancers or promoters, as well as real-time imaging of chromatin structure, the research group will study the effects of epigenetic marks on the dynamic 3D structure of chromatin.

Project 2.2: In vivo Sequence Sensors and Intelligent Therapies Based on Synthetic Biological Circuits

FIG 3. Study the causal relationship between epigenetics, three-dimensional genome structuredynamics, and cell phenotypes through Casilio epigenetic editing and real-time in vivo genome imaging.

This project aims to develop sequence sensors that can work in real-time in live cells. By coupling these sensors to synthetic biological response circuits, specific cellular programs can be triggered when detecting certain DNA or RNA sequences, achieving therapeutic functions. For example, inducing malignant cell self-destruction specifically upon detecting specific genetic mutations. In the preliminary work, the research group has already used TALE or CRISPR/Cas as DNA recognition modules to generate these sensors. When two DNA binding domains closely bind together at the target site, protein splicing recombination occurs based on proximity-induced intein, forming a transducing transcription factor. The transducing transcription factor will bind to the response circuit and trigger the expression of a transgene (such as a toxin), achieving specific effects. Next, DNA sensors will be designed to identify cancer-specific fusion genes or mutations, and toxin or prodrug genes will be used as response genes to test the synthetic biological circuit's efficacy against cancer-specific cells. The research group will further develop in vivo sequence sensors and cell state sensors, develop and apply synthetic biology logic gates to connect these sensors to Casilio and ACRE, to achieve "smart gene therapy". This novel gene therapy will provide significant breakthroughs for precision and personalized treatment.

FIG 4. Combine sequence and cell state sensors, biological logic gates, and writing systems to construct synthetic biological circuits that respond to cellular states and genotypes, producing different gene therapy effects.

Selected publications:

*Corresponding, #co-first

1. Liu, Z., Jillette N., Robson, P., Cheng, A.W.* (2023) Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold. Nucleic Acid Research gkad547 doi: 10.1093/nar/gkad547. IF=19.16
2. Clow, P.A., Du, M., Jillette, N., Taghbalout, A., Zhu, J.J.*, Cheng, A.W.* (2022) CRISPR-mediated multiplexed live cell imaging of nonrepetitive genomic loci with one guide RNA per locus. Nature Communications 13:1871. doi: 10.1038/s41467-022-29343-z. IF=17.694
3. Yi, E., Gujar, A.D., Guthrie, M., Kim, H., Zhao, D., Johnson K.C., Amin, S.B., Costa, M.L., Yu, Q., Das, S., Jillette, N., Clow, P.A., Cheng, A.W.*, Verhaak, R.G.W.* (2022) Live-cell imaging shows uneven segregation of extrachromosomal DNA elements and transcriptionally active extrachromosomal DNA hubs in cancer. Cancer Discovery doi: 10.1158/2159-8290.CD-21-1376 (co-corresponding) IF=39.397
4. Zhu, J.J., Cheng, A.W.* (2022) JACKIE: Fast enumeration of genome-wide single- and multi-copy CRISPR target sites and their off-target numbers. The CRISPR Journal doi: 10.1089/crispr.2022.0042 IF=4.321
5. Du, M.#, Jillette, N.#, Zhu, J.J., Li, S., Cheng, A.W.* (2020) CRISPR Artificial Splicing Factors. Nature Communications 11:2973. doi: 10.1038/s41467-020-16806-4 IF=17.694
6. Zhu, J.J., Jillette, N., Li X., Cheng, A.W.*, Lau C.C.* (2020) C11orf95-RELA Reprograms 3D Epigenome in Supratentorial Ependymoma. Acta Neuropathologica doi: 10.1007/s00401-020-02225-8 (co-corresponding) IF=21.534
7. Taghbalout, A., Du, M., Jillette, N., Rosikiewicz, W. Rath, A., Heinen, C., Li, S., Cheng, A.W.* (2019) Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways. Nature Communications 10:4296. doi:10.1038/s41467-019-12339-7 IF=17.694
8. Jillette, N.#., Du, M.#, Zhu, J.J., Cardoz, P., Cheng, A.W.* (2019) Split Selectable Markers. Nature Communications 10:4968. doi: 10.1038/s41467-019-12891-2 IF=17.694
9. Cheng, A.W.*#, Jillette, N.#, Lee, P., Plaskon, D., Fujiwara, Y., Wang, W., Taghbalout, A., Wang, H.* (2016) Casilio: a versatile CRISPR-Cas9-Pumilio hybrid for gene regulation and genomic labeling. Cell Research 26:254–257. doi: 10.1038/cr.2016.3 PMID:26768771 (co-corresponding) IF=46.3
10. Cheng, A.W.#, Shi, J.#, Wong, P.#, Luo, K.L., Trepman, P., Wang, E.T., Choi, H., Burge, C.B., Lodish, H.F.* (2014) Muscleblind-like 1 (Mbnl1) regulates pre-mRNA alternative splicing during terminal erythropoiesis. Blood doi: 10.1182/blood-2013-12-542209 PMID: 24869935 IF=25.48
11. Cheng, A.W.#, Wang, H.#, Yang, H, Shi, L., Katz, Y., Theunissen, T.W., Rangarajan, S., Shivalila, C.S., Dadon, D.B., Jaenisch, R.* (2013) Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Research 23(10):1163-71 PMID: 23979020 IF=46.3
12. Yang, H.#, Wang, H.#, Shivalila, C.S.#, Cheng, A.W., Shi, L., Jaenisch, R.* (2013). One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154(6):1370-9 PMID: 23992847
13. Wang, H.#, Yang, H.#, Shivalila, C.S.#, Dawlaty, M.M., Cheng, A.W., Zhang, F., Jaenisch, R.* (2013). One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153(4):910-8 PMID: 23643243
14. Buganim, Y.#, Faddah D.A.#, Cheng, A.W., Itskovich, E., Markoulaki, S., Gantz, K., Klemm S.L., van Oudenaarden A., Jaenisch, R.* (2012) Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase. Cell 150(6):1209-22 PMID: 22980981
15. Shapiro, I.M.#, Cheng, A.W.#, Flytzanis, N.C., Balsamo, M., Condeelis, J.S., Oktay, M.H., Burge, C.B.*, Gertler, F.B.* (2011) An EMT-driven alternative splicing program occurs in human breast cancer and modulates cellular phenotype. PLoS Genet. 7(8):e1002218 PMID: 21876675 (co-first) IF=6.02
16. Creyghton, M.P.#, Cheng A.W.#, Welstead, G.G., Kooistra, T., Carey, B.W., Steine, E.J., Hanna, J., Lodato, M.A., Frampton, G.M., Sharp, P.A., Boyer, L.A., Young, R.A.*, Jaenisch, R.* (2010) Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. U.S.A. 107(50):21931-6 PMID: 21106759 (co-first) IF=12.78