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The effect of extracellular matrix stiffness on cell growth and the underlying molecular mechanism was investigated using an in situ single-cell imaging of gene expression method based on rolling circle amplification., Cell behaviors are known to be regulated by the cellular microenvironment. Traditional cell-population based analysis methods need to separate cells from their extracellular matrix (ECM) and cannot resolve the heterogeneity of cell behaviors. Herein, an in situ single-cell analysis method based on rolling circle amplification was exploited to image gene expression in single cells for investigating the effect of ECM stiffness on cell growth. This method enables the simultaneous quantifying of the cell phenotype and gene expression at the single-cell level, which can help in understanding the underlying molecular mechanism of cell growth. It is found that ECM stiffness could affect cell growth via regulating the expression level of the cytoskeleton-assembly associated genes PFN1 and CFL1 and their co-expression pattern. Therefore, this single-cell analysis platform may facilitate us to tap into the study of “single-cell phenotypes” and elucidate the disease association of ECMs.

DNA nano-spring multivalent ligands for reversibly controlling the nanoscale distribution of cell binding ligands and regulating the cell behavior., Chemically functionalized and nanostructured materials, which mimic the features of the natural extracellular matrix, provide a tool to organize cell surface receptors into nanoscale clusters and manipulate cell functions. However, the existing materials are mainly based on static structures. Herein, we developed a DNA based structure-switchable and multivalent material that acts as a ‘nano-spring’, enabling reversible control of membrane receptor function at the cell surface. This ‘nano-spring’ can be easily synthesized by rolling circle amplification and finely tuned by changing the circular template design. Using this ‘nano-spring’ to interact with cells, we have demonstrated that the movement of the DNA nanostructure is sufficient to direct a cell morphology change from the normal morphology to having numerous cell protrusions and affect the mRNA expression level of integrin related genes. This DNA nano-spring structure can be a competitive material for actively manipulating cell receptor function and may help us to understand the role of receptor mediated signalling cascades.

We report a robust method for the efficient imaging of mRNA with single-nucleotide and near-single-molecule resolution in single cells., Detection of single-cell gene expression with high spatial and sequence resolution is a key challenge in single cell biology. Herein, we propose a robust method for the direct detection of mRNA, termed target RNA-initiated rolling circle amplification, which enables imaging of mRNA with single-nucleotide and near-single-molecule resolution in single cells. By utilizing a Splint R ligase capable of efficiently catalyzing the ligation of a padlock probe by the target RNA, the method can enable the efficient detection of mRNA without reverse transcription (detection efficiency over 20%). Meanwhile, attributed to the ligation-based recognition process, the method confers specificity sufficient to genotype mRNAs with one-nucleotide variations. The method has enabled the spatial mapping and correlation analysis of gene expression in single cells which could help us to elucidate the gene functions and regulatory mechanisms. This method offers an mRNA profiling ability with high spatial resolution and sequence specificity, thus is expected to be a single-cell analysis platform for not only investigating gene expression, but also potentially for analyzing single-nucleotide variants or mRNA alternative splicing at single-cell level.

A novel Cas9 cleavage assay was developed for quantitative evaluation of Cas9 cleavage efficiency and pre-screening of sgRNA to achieve highly specific and highly efficient CRISPR/Cas9 genome editing., CRISPR/Cas9 is a highly efficient genome engineering tool, yet its off-target effects and sequence-dependent cleavage activity across different sgRNAs remain major concerns for its application. Here, we propose a nicking triggered exponential amplification reaction (NTEXPAR), a fast and sensitive in vitro method, to detect the double strand DNA cleaved by down to 10 pM Cas9 with a linear range of 100 pM to 20 nM. With this newly developed amplification method, Cas9 cleavage activity can be quantified in 40 min and the optimal sgRNA design for specific target sequence can be successfully determined. Using the pre-screened sgRNA, we are able to distinguish single nucleotide mismatch in a gene silencing experiment. This fluorescence based isothermal assay provides a versatile tool for the pre-screening of sgRNAs to achieve highly specific and highly efficient CRISPR/Cas9 genome editing.