A super sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair, bioRxiv, 2020-01-21
AbstractAuxin-Inducible Degron (AID) technology enables conditional depletion of targeted proteins. However, the applicability of the AID in vertebrate cells has been limited due to cytotoxicity caused by high auxin concentrations. Here, we establish an improved AID system using an engineered orthogonal auxin-TIR1 pair, which exhibits over 1,000 times stronger binding. With ~1,000-fold less auxin concentration, we achieved to generate the AID-based knockout cells in various human and mouse cell lines in a single transfection.
biorxiv bioengineering 0-100-users 2020On-demand spatiotemporal programming of collective cell migration via bioelectric stimulation, bioRxiv, 2019-12-23
Directed cell migration is critical across biological processes spanning healing to cancer invasion, yet no tools allow such migration to be interactively guided. We present a new bioreactor that harnesses electrotaxis—directed cell migration along electric field gradients—by integrating multiple independent electrodes under computer control to dynamically program electric field patterns, and hence steer cell migration. Using this platform, we programmed and characterized multiple precise, two-dimensional collective migration maneuvers in renal epithelia and primary skin keratinocyte ensembles. First, we demonstrated on-demand, 90-degree collective turning. Next, we developed a universal electrical stimulation scheme capable of programming arbitrary 2D migration maneuvers such as precise angular turns and directing cells to migrate in a complete circle. Our stimulation scheme proves that cells effectively timeaverage electric field cues, helping to elucidate the transduction time scales in electrotaxis. Together, this work represents a fundamentally different platform for controlling cell migration with broad utility across fields.
biorxiv bioengineering 200-500-users 2019Biological Plasticity Rescues Target Activity in CRISPR Knockouts, bioRxiv, 2019-07-27
AbstractGene knockouts (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While DNA editing efficiency is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here, we devised an experimental strategy combining RNA-seq and triple-stage mass spectrometry (SPS-MS3) to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins, or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9 generated KO lines is necessary for phenotype interpretation.
biorxiv bioengineering 100-200-users 2019μDamID a microfluidic approach for imaging and sequencing protein-DNA interactions in single cells, bioRxiv, 2019-07-19
AbstractGenome regulation depends on carefully programmed protein-DNA interactions that maintain or alter gene expression states, often by influencing chromatin organization. Most studies of these interactions to date have relied on bulk methods, which in many systems cannot capture the dynamic single-cell nature of these interactions as they modulate cell states. One method allowing for sensitive single-cell mapping of protein-DNA interactions is DNA adenine methyltransferase identification (DamID), which records a protein’s DNA-binding history by methylating adenine bases in its vicinity, then selectively amplifies and sequences these methylated regions. These interaction sites can also be visualized using fluorescent proteins that bind to methyladenines. Here we combine these imaging and sequencing technologies in an integrated microfluidic platform (μDamID) that enables single-cell isolation, imaging, and sorting, followed by DamID. We apply this system to generate paired single-cell imaging and sequencing data from a human cell line, in which we map and validate interactions between DNA and nuclear lamina proteins, providing a measure of 3D chromatin organization and broad gene regulation patterns. μDamID provides the unique ability to compare paired imaging and sequencing data for each cell and between cells, enabling the joint analysis of the nuclear localization, sequence identity, and variability of protein-DNA interactions.
biorxiv bioengineering 0-100-users 2019Octopi Open configurable high-throughput imaging platform for infectious disease diagnosis in the field, bioRxiv, 2019-06-28
AbstractAccess to quantitative, robust, yet affordable diagnostic tools is necessary to reduce global infectious disease burden. Manual microscopy has served as a bedrock for diagnostics with wide adaptability, although at a cost of tedious labor and human errors. Automated robotic microscopes are poised to enable a new era of smart field microscopy but current platforms remain cost prohibitive and largely inflexible, especially for resource poor and field settings. Here we present Octopi, a low-cost ($250-$500) and reconfigurable autonomous microscopy platform capable of automated slide scanning and correlated bright-field and fluorescence imaging. Being highly modular, it also provides a framework for new disease-specific modules to be developed. We demonstrate the power of the platform by applying it to automated detection of malaria parasites in blood smears. Specifically, we discovered a spectral shift on the order of 10 nm for DAPI-stained Plasmodium falciparum malaria parasites. This shift allowed us to detect the parasites with a low magnification (equivalent to 10x) large field of view (2.56 mm2) module. Combined with automated slide scanning, real time computer vision and machine learning-based classification, Octopi is able to screen more than 1.5 million red blood cells per minute for parasitemia quantification, with estimated diagnostic sensitivity and specificity exceeding 90% at parasitemia of 50ul and 100% for parasitemia higher than 150l. With different modules, we further showed imaging of tissue slice and sputum sample on the platform. With roughly two orders of magnitude in cost reduction, Octopi opens up the possibility of a large robotic microscope network for improved disease diagnosis while providing an avenue for collective efforts for development of modular instruments.One sentence summaryWe developed a low-cost ($250-$500) automated imaging platform that can quantify malaria parasitemia by scanning 1.5 million red blood cells per minute.
biorxiv bioengineering 500+-users 2019Optogenetic control of Wnt signaling for modeling early embryogenic patterning with human pluripotent stem cells, bioRxiv, 2019-06-10
ABSTRACTThe processes of cell proliferation, differentiation, migration, and self-organization during early embryonic development are governed by dynamic, spatially and temporally varying morphogen signals. Analogous tissue patterns emerge spontaneously in embryonic stem cell (ESC) models for gastrulation, but mechanistic insight into this self-organization is limited by a lack of molecular methods to precisely control morphogen signal dynamics. Here we combine optogenetic stimulation and single-cell imaging approaches to study self-organization of human pluripotent stem cells. Precise control of morphogen signal dynamics, achieved through activation of canonical Wntβ-catenin signaling over a broad high dynamic range (>500-fold) using an optoWnt optogenetic system, drove broad transcriptional changes and mesendoderm differentiation of human ESCs at high efficiency (>95% cells). Furthermore, activating Wnt signaling in subpopulations of ESCs in 2D and 3D cultures induced cell self-organization and morphogenesis reminiscent of human gastrulation, including changes in cell migration and epithelial to mesenchymal transition. Our findings thus reveal an instructive role for Wnt in directing cell patterning in this ESC model for gastrulation.
biorxiv bioengineering 0-100-users 2019