We are interested in developing novel technologies of genetic engineering, broadly defined, for controlling, manipulating, and engineering the genetic codes of life. We believe the ability to controlling the genome is of fundamentally importance to understanding its function in physiology and disease, which provides a foundation for rationally designing and engineering the genome and cells for diagnosis and therapeutics. We regard such an approach as discovery-based synthetic biology.
Towards this goal, we have developed the nuclease-deactivated Cas9 (dCas9) molecule from the prokaryotic CRISPR system for multiple purposes: CRISPRi/a for transcription activation or repression, genome imaging for genome tracking in living cells, and CRISPR-GO for manipulating the 3D genome organization in the nucleus. Meantime, we appreciate the vast macromolecules (DNA, RNA, and proteins) that have been evolved from billion years history, and aim to harness these naturally occurring molecules as tools. We also appreciate the power of directed evolution to obtain novel molecules that have been discovered or evolved in Nature. We feel there is a responsibility and a need to harness technology and engineering for studying biology to promote science and the society. Three topics are particularly interesting to us:
- New molecules as tools and technologies for engineering human genetics, broadly defined.
- An integrated mammalian genome: how sequence, genes, regulatory elements, and 3D structure interplay to maintain and execute functions.
- Rational design of genetic systems and the whole cell for immunological engineering and regenerative medicine.
New molecules as tools and technologies for engineering human genetics, broadly defined Example: dCas9; CRISPRi/a; CRISPR imaging; CRISPR-GO
The CRISPR-Cas nucleases have made genome editing several orders of magnitude easier in diverse organisms. Beyond editing, we developed the nuclease-dead Cas9 (dCas9) molecule for sequence-specific DNA binding. We demonstrated the use of the dCas9 for gene regulation, genome imaging, and 3D genome structural control. For gene regulation, we developed CRISPRi (interference, Qi Cell 2013), the first demonstration of dCas9 in cells. Working with our collaborator, Wendell Lim, Jonathan Weissman and Jennifer Doudna, we developed expanded CRISPRi to CRISPRa (activation, Gilbert Cell 2013) in yeast and mammalian cells. For genome imaging, we co-developed using CRISPR-dCas9 fused to GFP for tracking dynamics of the genome locus in living cells (Chen Cell 2013) with Bo Huang’s lab. For 3D genome control, we developed CRISPR-GO (genome organization, Wang Cell 2013) for repositioning DNA of interest to the nuclear periphery, Cajal bodies, and PML bodies. We improved the tools for multiplexed gene expression control (Zalatan Cell 2015; Gao Nature Methods 2016), combined CRISPR with chemical or light-inducible systems (Gao Nature Methods 2016; Kipniss Nature Communications 2017), and built genetic circuits based on the CRISPR-dCas9 systems. The goal of tool development is to control any genes (coding, noncoding) for any mode (transcription, epigenetic, spatial, temporal, multiplexed, quantitative, inducible, …) in any cell (mitotic, non-mitotic). Examples include:
· First development of the dCas9 molecule and CRISPRi (Qi Cell 2013)
· Development of CRISPRi/a in mammalian cells (Gilbert Cell 2013)
· Inducible and multiplexed gene regulation (Zalatan Cell 2015; Gao Nature Methods 2016)
· CRISPRI/O: Putting CRISPR under the control of cellular receptor ligands (Kipniss Nature Communication 2017)
· CRISPR imaging for tracking the dynamics of genome in real-time (Chen Cell 2013)
· CRISPR-GO: manipulating the 3D genome structure within the nucleus (Wang Cell 2018)
· We teamed up with a number of labs (Wendell Lim, Jennifer Doudna, Jonathan Weissman, Ron Vale, Carol Gross) and demonstrated how the CRISPRi/a system can be used to study functional genomics in bacteria, yeast, and mammalian cells.