My research broadly aims to develop CRISPR-Cas-based tools for gene regulation. Historically, my work has focused on developing tools which allow for physically repositioning genes to the nuclear periphery or the formation of artificial DNA loops. Currently, I am focusing on developing logic gates for complex genetic circuit construction in yeast. 

CRISPR-Based Genetic Logic Gates

Synthetic biologists are seeking to create new biological parts and systems for applications to fields such as metabolic engineering or bio-manufacturing. Logic gates have been a long standing model for our understanding of how gene expression programs function and can be constructed. Logic gates are the rational combination of one or more inputs to produce an output. To date, sophisticated circuits have been developed in yeast that allow for the construction of many forms of logic (Gander, 2017.). However, as we seek to develop increasingly complex genetic logic systems, we need to simplify how we approach the construction of some forms of logic (Figure 1). To address this need, I have developed single-layer AND and NAND genetic logic gates in yeast (Figure 2). 

Figure 1. AND and NAND gates constructed from a circuit of NOR gates are the current standard for the formation of AND and NAND genetic logic. I have developed single-layer AND and NAND gates. This should allow for the development of more complex genetic logic systems with simplified circuits.

Figure 2. The AND and NAND gate systems utilize the co-LOCKR system to recruit an activator or repressor upstream of a reporter gene. The activator or repressor will only be localized upstream of the reporter gene if both the KEY and CAGE components of the co-LOCKR system are present. This is the foundation for AND functionality.

CRISPR-Based Gene Peripheral Recruitment

Previous studies have demonstrated that genes positioned near the nuclear periphery tend to be in a repressed state and genes positioned in the nuclear interior tend to be in an active state (Egecioglu 2011, Ramani 2016, Misteli 2020). Furthermore, genes can be dynamically repositioned upon activation or in response to extracellular signals (Casolari 2004, Brickner 2004, Randise-Hinchliff 2016, Kim 2017).

To test the functional effects of peripheral gene localization on gene expression levels, I developed a programmable CRISPR-Cas system for nuclear peripheral recruitment in yeast (Figure 3) (Cliff 2021). I benchmarked this system in yeast using microscopy and gene silencing assays and have demonstrate that CRISPR-Cas-mediated tethering can recruit some loci, but not all. Additionally, CRISPR-Cas-mediated recruitment was not demonstrated to detectably silence reporter gene expression. 

Current work on this project is being done in collaboration with the Bertero and Disteche labs. 

For my ongoing collaboration with the Bertero group, we have been working to implement our CRISPR-Cas-mediated gene relocalization tool in hiPSCs (human induced pluripotent stem cells) to explore the causal role of gene localization in cellular differentiation, particularly for human cardiomyocytes. 

I also have an ongoing collaborative effort with the Disteche Lab. For this project, we have been working to implement the Qi Lab’s CRISPR-GO tool for gene repositioning in PATSKI cells (embryonic mouse kidney fibroblast cells) for the study of X-chromosome inactivation (Wang 2019).  

 Figure 3. Genomic sites can be physically repositioned by fusing a DNA binding domain to a membrane protein. The DNA binding domain in this instance is a CRISPR-Cas complex that can be programmed to different target sites. The CRISPR-Cas complex can be linked to a membrane protein via a scaffold RNA (scRNA), a modified gRNA that includes an MS2 RNA hairpin to recruit the MS2 coat protein (MCP) (Zalatan 2015). MCP is fused to the membrane protein, thus recruiting the gene to the nuclear membrane. 

CRISPR-Based Synthetic Gene Looping

Studies have demonstrated that chromatin loop structures play a role in gene regulation. These structures can range in size and form from trans-acting activators and repressors to large topologically associating domains (TADs, averaging 1MB and ~10 genes), wherein genes typically maintain similar states of expression (Dekker 2015, Halfon 2020, Vermunt 2019, Lieberman-Aiden 2009, Franke 2016). In contrast, genome structures can undergo major perturbations with only modest effect on the transcriptome when cohesin-mediated loops are disrupted in human cells (Rao 2017).

To better understand the influence of looping on gene regulation, synthetic gene looping systems have been developed (Priest 2013, Morgan 2017, Hao 2021). In these systems, a protein consisting of a DNA-binding domain and dimerization domain bind DNA and each other to bring two sections of DNA together (Figure 4, upper). However, these systems suffer from inefficient looping, particularly over longer genomic distances. A major factor contributing to this inefficiency is binding of DNA-bound complexes by free complexes, which can then weaken or block loop formation (Figure 4, upper) (Priest 2013).  

 Figure 4. When creating synthetic loops, binding to free complexes competes with loop formation (Priest 2013). We hypothesis that we can more effectively create synthetic DNA loops by building a system that only activates once the components have bound DNA. 

One potential solution to this problem is an allosteric sensor of DNA binding. This system would only “activate” or “open” it’s dimerization domain once it has bound DNA (Figure 4, lower). Utilizing the LOCKR system, we have developed a synthetic switch which only activates once it has bound DNA (Kirkpatick 2019). I am currently working to apply this synthetic switch to create a LOCKR-based DNA looping system (Figure 5). In this system, looping can only occur if the latch is opened via co-localization of a “key” peptide. This frees the dimerization motif to bind its partner. I have promising preliminary results for two sets of dimerization domains and am working to form synthetic loops in bacterial and human cell models. 

Much of the development for caged heterodimers on this project is being done in collaboration with the Baker Lab. 

 Figure 5. A LOCKR-based looping system. This system combines CRISPR-Cas technology for DNA binding with the LOCKR system (Langan 2019). The LOCKR system is a de novo designed protein switch. The system “cages” a protein so that it is not accessible unless the “key” is present to unlock the cage. Unlocking the cage allows the caged heterodimer protein on the latch (blue coil) to bind its partner (other blue coil) in the above diagram. Thus, the system restricts the binding of free complex and should improve DNA looping efficiency. 

Citations

Brickner, J. H., and Walter, P. Gene recruitment of the activated INO1 locus to the nuclear membrane. PLoS Biol. 2, e342 (2004). 

Cliff, E. R. et al. CRISPR-Cas-Mediated Tethering Recruits the Yeast HMR Mating-Type Locus to the Nuclear Periphery but Fails to Silence Gene Expression. ACS Synth. Biol. 10, 2870–2877 (2021). 

Casolari, J. M. et al. Genome-wide localization of the nuclear transport machinery couples transcriptional status and nuclear organization. Cell 117, 427–439 (2004). 

Dekker, J. & Misteli, T. Long-Range Chromatin Interactions. Cold Spring Harb Perspect Biol 7, a019356 (2015). 

Egecioglu, D. and Brickner, J. H. Gene positioning and expression. Curr. Opin. Cell Biol. 23, 338–345 (2011). 

Franke, M. et al. Formation of new chromatin domains determines pathogenicity of genomic duplications. Nature 538, 265–269 (2016). 

Gander, M. W., Vrana, J. D., Voje, W. E., Carothers, J. M. & Klavins, E. Digital logic circuits in yeast with CRISPR-dCas9 NOR gates. Nat Commun 8, 15459 (2017).

Halfon, M. S. Silencers, Enhancers, and the Multifunctional Regulatory Genome. Trends in Genetics 36, 149–151 (2020). 

Hao, N., Shearwin, K. E. & Dodd, I. B. Programmable DNA looping using engineered bivalent dCas9 complexes. Nature Communications 8, 1628 (2017). 

Kim, S. et al. The dynamic three-dimensional organization of the diploid yeast genome. eLife 6 (2017). 

Kirkpatrick, R. L. et al. Conditional Recruitment to a DNA-Bound CRISPR–Cas Complex Using a Colocalization-Dependent Protein Switch. ACS Synth. Biol. 9, 2316–2323 (2020). 

Langan, R. A. et al. De novo design of bioactive protein switches. Nature 572, 205–210 (2019). 

Lieberman-Aiden, E. et al. Comprehensive Mapping of Long-Range Interactions Reveals Folding Principles of the Human Genome. Science 326, 289–293 (2009). 

Misteli, T. The self-organizing genome: Principles of genome architecture and function. Cell 183, 28–45 (2020). 

Morgan, S. L. et al. Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping. Nature Communications 8, 15993 (2017). 

Priest, D. G. et al. Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors. PNAS (2013) doi:10.1073/pnas.1317817111. 

Ramani, V. et al. Understanding spatial genome organization: methods and insights. Genomics Proteomics Bioinformatics 14, 7–20 (2016). 

Randise-Hinchliff, C., and Brickner, J. H. Transcription factors dynamically control the spatial organization of the yeast genome. Nucleus 7, 369–374 (2016). 

Rao, S. et al. Cohesin loss eliminates all loop domains. Cell 171, 305–320.e24. (2017). 

Vermunt, M. W., Zhang, D. & Blobel, G. A. The interdependence of gene-regulatory elements and the 3D genome. Journal of Cell Biology 218, 12–26 (2018). 

Wang, H. et al. CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization. Cell 175, 1405-1417.e14 (2018). 

Zalatan, J. G. et al. Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell  160, 339–350 (2015).