(6kh) Rewritable Multi-event Analog Recording in Bacteria and Mammalian Cells

Stable recording of cellular events can significantly advance our understanding of a cell’s history and its responses to stimuli. However, the construction of synthetic memory devices, especially ones with large storage capacities, remains challenging. We recently developed two platforms that use base editors and CRISPR-Cas9 nucleases to stably record molecular events of interest, including exposure to antibiotics, nutrients, viruses, and light, in living cells. Reliable readout can be obtained by sequencing as few as dozens of cells and the formed synthetic memories can be erased for repeated recording. Moreover, the base editor-mediated system functions in human cells, recording the presence of exogenous small molecules as well as changes in endogenous Wnt signaling in a safe-harbor locus of the human genome. We expect these CRISPR-mediated analog multi-event recording apparatus (CAMERA) systems to serve as “cell data recorders” that write a history of endogenous or exogenous signals into permanent DNA sequence modifications in living cells.

Postdoctoral Project: engineering the CRISPR system for broadened applications

Postdoctoral Fellowship: the Jane Coffin Childs Memorial Fund

Supervised by David R. Liu, Broad Institute of MIT and Harvard; Department of Chemistry and Chemical Biology, University of Harvard

PhD Dissertation: mechanistic studies of lanthipeptide biosynthesis

Supervised by Wilfred A. van der Donk, Chemical, Department of Chemistry, University of Illinois, Urbana-Champaign

Research Interests:

As a graduate student in Prof. van der Donk’s lab, I focused on uncovering the biosynthetic mechanisms of lanthipeptides, a family of ribosomal natural products with extensive post-translational modifications. Using recombinant expression systems, I successfully prepared six lanthipeptides that could not be obtained from native producers and characterized their structures by NMR. During my study of cytolysin, an important virulence factor produced by Enterococcus faecalis, I discovered an unusual lanthionine stereochemistry that was different from all documented lanthionine structures in lanthipeptides. I further confirmed that, surprisingly, the formation of the unusual stereochemistry was controlled by a substrate motif rather than the synthetase. Following my discovery, more than a dozen other lanthipeptides have been reported to contain this substrate-determined LL stereochemistry.

After graduation, I moved to a different research area and worked on programmable genome-editing agents focusing on the CRISPR system in Prof. Liu’s lab. In collaboration with graduate students in the Liu lab, I engineered Cas9 variants for expanded protospacer adjacent motif (PAM) specificities, enabling Cas9-mediated activities to be directed to a significantly expanded variety of genomic loci. In a second project, I took a synthetic biologist’s perspective and developed a set of memory devices using the CRISPR technology to facilitate faithful and durable recording of stimuli and cell state changes in bacteria including exposure to antibiotics, nutrients, viruses, and light. These devices can also function in mammalian cells to record the activity fluctuation of critical endogenous signaling pathways.

Working on various research topics and interacting with scientists from different technical backgrounds have made me appreciate the advantages of a well-diversified research program, and have inspired me to pursue a wide variety of research projects in my own group.

Teaching Interests:

My experiences in science have made me passionate about teaching. I served as a teaching assistant in general chemistry classes at the University of Illinois, Urbana-Champaign and have mentored several Master’s and junior PhD students both in graduate school and as a postdoctoral researcher. To better prepare myself for the upcoming teaching responsibility, I attended the Kaufman Teaching Certificate Program at MIT during the spring semester of 2018. In this 12-week program, I improved a variety of my teaching skills, including designing a course syllabus, interactive teaching and active learning, designing assignments and problem sets, and how to teaching inclusively.

Given my dual background in chemistry and biology, I can teach a variety of undergraduate courses such as General Chemistry, Biochemistry, and their lab sessions as well as graduate-level courses such as Recent Advances in Chemical Biology, Synthetic Biology, Mechanistic Enzymology, and Modern Therapeutics in the Genomic Era.

Future Direction

As an assistant professor, I aim to utilize my expertise in chemical biology and bioengineering to develop approaches that enable the investigation of three critical life science questions: 1) are novel DNA base modifications awaiting discovery? 2) can highly functionalized cyclic peptides provide improved pharmacokinetic properties and realize the ultimate therapeutic potential of peptide natural products? 3) can cell state dynamics be recorded by taking full advantage of the programmable nature of DNA?

More than 10 types of nucleotide thiolation have been reported in RNA, yet no such modification has been found in DNA, likely owing to the lack of characterization approaches. In my first project, I aim to develop sequencing techniques to read out sulfur-modifications in DNA at base resolution. These sequencing techniques will not only facilitate the investigation of the native presence of these thionucleosides in DNA, but also enable the application of sulfur-modified deoxyribonucleotide as DNA labeling probes to study genome dynamics at a heretofore inaccessible resolution and depth.

Cyclic peptides are better drug candidates than linear peptides because they offer better target binding affinity and specificity as well as improved pharmacokinetic properties. However, current strategies of synthesizing cyclic peptide libraries often provide limited complexity and are not compatible with high throughput screenings due to the lack of genotype-phenotype correlation. In my second project, I aim to construct highly functionalized cyclic peptide libraries by deriving a family of peptide natural products that are genetically encoded and post-translationally modified. A microfluidics-enabled selection platform will be engineered to screen for cyclic peptides that bind and regulate target proteins in live cells.

In my third project, I plan to engineer a targeted in situ DNA diversification tool that has broad applications ranging from construction of complex synthetic memory that facilitates single-cell lineage tracing to focused randomization of genes of interest in live cells for forward genetic studies and continuous directed evolution.

The proposed projects will not only use chemical biology principles to push the boundaries of science in areas of epigenetics, peptide drug design, and cell state dynamic tracking, but will also facilitate many future studies by offering valuable tools to efficiently label DNA with probes that can be mapped at base resolution, synthesize cyclic peptide libraries of high diversity and complexity, screen large compound libraries carried by live cells in therapeutic-enabling environments, and diversify the target DNA without affecting other sequences during directed evolution.

Selected Publications

Tang W; Liu DR*. Rewritable multi-event analog recording in bacterial and mammalian cells. Science 2018, 360, eaap8992

Tang W; Hu JH; Liu DR*. Aptazyme-embedded guide RNAs enable ligand-responsive genome engineering. Nat. Commun. 2017, 8, 15939

Tang W; Thibodeaux GN; van der Donk WA*. The enterococcal cytolysin synthetase coevolves with substrate for stereoselective lanthionine synthesis. ACS Chem. Biol. 2016, 11, 2438-2446.

Dong SH^#, Tang W^#, Lukk T, Nair SK*, van der Donk WA*. The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold. eLife 2015, 4, 07607 (# equal contribution)

Tang W, Jiménez-Osés G, Houk KN*, van der Donk WA*. Substrate control in stereoselective lanthionine biosynthesis. Nat. Chem. 2015, 7, 57-64

Tang W^#, Dong SH^#, Repka LM, He C, Nair SK*, van der Donk WA*. Applications of the class II lanthipeptide protease LicP for sequence-specific, traceless peptide bond cleavage. Chem. Sci. 2015, 6, 6270-6279 (# equal contribution)

Tang W, van der Donk WA*. The sequence of the enterococcal cytolysin imparts unusual lanthionine stereochemistry. Nat. Chem. Biol. 2013, 9, 157-159

Tang W, van der Donk WA*. Structural characterization of four prochlorosins: a novel class of lantipeptides produced by planktonic marine cyanobacteria. Biochemistry 2012, 51, 4271-4279

Patents

van der Donk WA, Garg N, Goto Y, Tang W. Class I and II lantibiotics from Geobacillus thermodenitrificans. U.S. Patent 9,326,523, issued on May 3, 2016; WIPO Pat. Appl. WO/2013/119821 A1, filed on August 15, 2013.

van der Donk WA, Tang W. Higher performance proteases for scarless tag removal. US-2017-0240878, published on August 24, 2017; WIPO Pat. Appl. WO/2015/175576 A3, filed on March 17, 2016.

Liu DR, Tang W, Hu JH. Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation. U.S. 62/505,175, provision filed on May 12, 2017.

Liu DR, Tang W. Cell data recorders and uses thereof. U.S. 62/631,455, provision filed on February 15, 2018.