Biology subjects

Qi, L. S.

Publications and source records attributed to Qi, L. S..

29 records · Page 2Linked to original sources

bioRxiv2021

Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus within 20 years that gave rise to a life-threatening disease and the first to reach pandemic spread. To make therapeutic headway against current and future coronaviruses, the biology of coronavirus RNA during infection must be precisely understood. Here, we present a robust and generalizable framework combining high-throughput confocal and super-resolution microscopy imaging to study coronavirus infection at the nanoscale. Employing the model human coronavirus HCoV-229E, we specifically labeled coronavirus genomic RNA (gRNA) and double-stranded RNA (dsRNA) via multicolor RNA-immunoFISH and visualized their localization patterns within the cell. The exquisite resolution of our approach uncovers a striking spatial organization of gRNA and dsRNA into three distinct structures and enables quantitative characterization of the status of the infection after antiviral drug treatment. Our approach provides a comprehensive framework that supports investigations of coronavirus fundamental biology and therapeutic effects.

cell biology↗

bioRxiv2021

Interrogation of the Dynamic Properties of Higher-Order Heterochromatin Using CRISPR/dCas9

Eukaryotic chromosomes feature large regions of compact, repressed heterochromatin hallmarked by Heterochromatin Protein 1 (HP1). HP1 proteins play multi-faceted roles in shaping heterochromatin, and in cells, HP1 tethering to individual gene promoters leads to epigenetic modifications and silencing. However, emergent properties of HP1 at supranucleosomal scales remain difficult to study in cells due to lack of appropriate tools. Here, we develop CRISPR-Engineered Chromatin Organization (EChO), combining live cell CRISPR imaging with inducible large-scale recruitment of chromatin proteins to native genomic targets. We demonstrate that human HP1 tiling across kilobase-scale genomic DNA forms novel contacts with natural heterochromatin, integrates two distantly targeted regions, and reversibly changes chromatin from a diffuse to compact state. The compact state exhibits delayed disassembly kinetics and represses transcription across over 600 kilobases. These findings support a polymer model of HP1-mediated chromatin regulation and highlight the utility of CRISPR-EChO in studying supranucleosomal chromatin organization in living cells.

genomics↗

bioRxiv2021

Quantification of durable CRISPR-based gene silencing activity

Development of CRISPR-based technologies for regulating gene expression stands to provide novel methods for the study and engineering of biological behavior. New tools capable of inducing long-lasting changes in gene expression will increase the utility of these techniques, providing durable effects from one-time doses of reagents. We describe here a reporter system for quantifying the ability of CRISPR-based effectors to induce stable gene repression. We observe a continuous gradation of the ability of these effectors to silence gene expression, depending on the domain composition and configuration. We also report the creation of a single CRISPR protein capable of producing durable gene silencing. This assay should allow for the continued development of enhanced gene repression tools which will be useful in a wide array of biological research and engineering applications.

synthetic biology↗

bioRxiv2021

Enhanced Cas12a multi-gene regulation using a CRISPR array separator

The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple crRNAs. Here, we report that Cas12a array performance is hypersensitive to the GC content of crRNA spacers, as high-GC spacers can impair activity of the downstream crRNA. We analyzed naturally occurring CRISPR arrays and observed that repeats always contain an AT-rich fragment that separates crRNAs; we term this fragment a CRISPR separator. Inspired by this observation, we designed short, AT-rich synthetic separators (synSeparators) that successfully removed the disruptive effects between crRNAs. We demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously unknown feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

synthetic biology↗

bioRxiv2020

Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS

In the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on cellular phenotype upstream of genetic analysis, particularly for large and complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow for precise single-cell encapsulation into picoliter-scale double emulsion droplets compatible with high-throughput phenotyping via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying size and morphology as well as a heterogeneous cell mixture of a whole dissociated flatworm (5 - 25 m in diameter) within highly monodisperse double emulsions (35 m in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. High-throughput FACS phenotyping of cellular picoreactors has the potential to shift the landscape of single-cell droplet microfluidics by expanding the repertoire of current nucleic acid droplet assays to include functional screening. ABSTRACT FIGURE O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/139311v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@9d2028org.highwire.dtl.DTLVardef@1aa55b3org.highwire.dtl.DTLVardef@1e05eb3org.highwire.dtl.DTLVardef@1695a88_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza

The outbreak of the coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), has infected more than 100,000 people worldwide with over 3,000 deaths since December 2019. There is no cure for COVID-19 and the vaccine development is estimated to require 12-18 months. Here we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells), for viral inhibition that can effectively degrade SARS-CoV-2 sequences and live influenza A virus (IAV) genome in human lung epithelial cells. We designed and screened a group of CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs for cleaving SARS-CoV-2. The approach is effective in reducing respiratory cell viral replication for H1N1 IAV. Our bioinformatic analysis showed a group of only six crRNAs can target more than 90% of all coronaviruses. The PAC-MAN approach is potentially a rapidly implementable pan-coronavirus strategy to deal with emerging pandemic strains.

bioengineering↗

bioRxiv2020

Systematic genome-wide querying of coding and non-coding functional elements in E. coli using CRISPRi

Genome-wide repression screens using CRISPR interference (CRISPRi) have enabled the high-throughput identification of essential genes in bacteria. However, there is a lack of functional studies leveraging CRISPRi to systematically explore targeting of both the coding and non-coding genome in bacteria. Here we perform CRISPRi screens in Escherichia coli MG1655 K-12 targeting ~13,000 genomic features, including nearly all protein-coding genes, non-coding RNAs, promoters, and transcription factor binding sites (TFBSs) using a ~33,000-member sgRNA library, which represents the most compact and comprehensive genome-wide CRISPRi library in E. coli to date. Our data reveal insights into the conditional essentiality of the genome with key refinements to screen design and profiling. First, we demonstrate that strong fitness defects associated with essential cellular processes can be resolved using inducible time-series measurements. We show that knockdowns of different classes of genes exhibit distinct, transient responses that are correlated to gene function with genes involved in translation exhibiting the strongest responses. We also query feature essentiality across several biochemical conditions and show that several genes, sRNAs, and operons exhibit conditional phenotypes not reported by previous high-throughput efforts. Second, we evaluate systematically targeting non-genic features (promoters and TFBSs) in the E. coli genome. We show that promoter-targeting guides can be used to add phenotypic confidence to promoter annotations and verify computationally predicted promoters. In contrast to prior studies, we find that promoter knockdowns exhibit a strong targeting orientation dependency where targeting the non-template strand of the promoter closest to the target gene is more effective in knocking down gene expression than other promoter targeting orientations. Unlike eukaryotic genomes, we note that interpreting the effects of TFBS targeting is particularly challenging due to the small size of such features and their proximity to and overlap with other genomic features. Together, this work reveals novel conditionally essential gene phenotypes, provides a characterized set of sgRNAs for future E. coli CRISPRi screens, and highlights considerations for CRISPRi library design and screening for microbial genome characterization.

microbiology↗

bioRxiv2019

Temporal-Spatial Visualization of Endogenous Chromosome Rearrangements in Living Cells

Visualizing the real-time dynamics of genome rearrangement in single living cells is core to studying genomics and diagnostics. Here, we report a robust, versatile approach named CRISPR Live-cell fluorescent in situ hybridization (LiveFISH) for multi-locus genome tracking and cytogenetic detection in a broad variety of cell types including primary cells. LiveFISH utilizes an intrinsic stability switch of CRISPR guide RNAs, which enables efficient and accurate detection of chromosomal disorders such as Patau Syndrome in prenatal amniotic fluid cells and allows multi-locus tracking in human T lymphocytes. Using LiveFISH, we are able to detect and track real-time spatiotemporal dynamics of non-homologous endogenous chromosome translocations induced by gene editing. This new approach enables FISH imaging in living primary cells, which can provide useful insights into the spatiotemporal changes of genome organization and rearrangements in normal and diseased primary cells and will enable fast cytogenetic visualization of various gene-editing associated chromosomal translocations.

bioengineering↗

bioRxiv2018

YAP-independent mechanotransduction drives breast cancer progression

Increased tissue stiffness is a driver of breast cancer progression. The transcriptional regulator YAP is considered a universal mechanotransducer, based largely on 2D culture studies. However, the role of YAP during in vivo breast cancer remains unclear. Here, we find that mechanotransduction occurs independently of YAP in breast cancer patient samples and mechanically tunable 3D cultures. Mechanistically, the lack of YAP activity in 3D culture and in vivo is associated with the absence of stress fibers and an order of magnitude decrease in nuclear cross-sectional area relative to 2D culture. This work highlights the context-dependent role of YAP in mechanotransduction, and establishes that YAP does not mediate mechanotransduction in breast cancer.\n\nOne Sentence SummaryBreast cancer mechanotransduction occurs independently of YAP, due to compact nuclear morphologies in vivo.

cancer biology↗

bioRxiv2017

Engineering Cell Sensing and Responses Using a GPCR-Coupled CRISPR-Cas System

G-protein coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes, and detects a wide array of physiological cues in the human body. We describe a new molecular device that couples CRISPR-Cas9 programmed genome regulation to natural and synthetic extracellular signals via GPCRs. The design of our synthetic device, named CRISPR ChaCha, displays superior performance over an architecture proposed by the previously reported Tango system. Using a parsimonious mathematical model and gene-reporter assays, we find that CRISPR ChaCha can recruit and activate multiple Cas9 molecules for each GPCR molecule. We also characterize key molecular features that modulate CRISPR ChaCha performance. We adopt the design to diverse GPCRs that sense synthetic and natural ligands including chemokines, mitogens, and fatty acids, and observe efficient conversion of signals to customizable genetic programs in mammalian cells, including regulation of endogenous genes. The new class of CRISPR-coupled GPCRs provides a robust and efficient platform for engineering cells with novel behaviors in response to the diverse GPCR ligand repertoire.

synthetic biology↗

bioRxiv2017

Multiplexed dynamic imaging of genomic loci in single cells by combined CRISPR imaging and DNA sequential FISH

Visualization of chromosome dynamics allows the investigation of spatiotemporal chromatin organization and its role in gene regulation and other cellular processes. However, current approaches to label multiple genomic loci in live cells have a fundamental limitation in the number of loci that can be labelled and uniquely identified. Here we describe an approach we call \"track first and identify later\" for multiplexed visualization of chromosome dynamics by combining two techniques: CRISPR labeling and DNA sequential fluorescence in situ hybridization (DNA seqFISH). Our approach first labels and tracks chromosomal loci in live cells with the CRISPR system, then barcodes those loci by DNA seqFISH in fixed cells and resolves their identities. We demonstrate our approach by tracking telomere dynamics, identifying 12 unique subtelomeric regions with variable detection efficiencies, and tracking back the telomere dynamics of respective chromosomes in mouse embryonic stem cells.

systems biology↗