Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.

Single-cell genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple omics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations (CNVs), DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs generally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells.

Circular RNAs (circRNAs) are a new class of non-polyadenylated non-coding RNAs that may play important roles in many biological processes. Here we develop a single-cell universal poly(A)-independent RNA sequencing (SUPeR-seq) method to sequence both polyadenylated and non-polyadenylated RNAs from individual cells. This method exhibits robust sensitivity, precision and accuracy. We discover 2891 circRNAs and 913 novel linear transcripts in mouse preimplantation embryos and further analyze the abundance of circRNAs along development, the function of enriched genes, and sequence features of circRNAs. Our work is key to deciphering regulation mechanisms of circRNAs during mammalian early embryonic development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0706-1) contains supplementary material, which is available to authorized users.

We combine fiber Bragg grating ͑FBG͒ technology with a wet chemical etch-erosion procedure and demonstrate two types of refractive index sensors using single-mode optical fibers. The first index sensor device is an etch-eroded single FBG with a radius of 3 m, which is used to measure the indices of four different liquids. The second index sensor device is an etch-eroded fiber Fabry-Pérot interferometer ͑FFPI͒ with a radius of ϳ1.5 m and is used to measure the refractive indices of isopropyl alcohol solutions of different concentrations. Due to its narrower resonance spectral feature, the FFPI sensor has a higher sensitivity than the FBG sensor and can detect an index variation of 1.4ϫ 10 −5 . Since we can measure the reflection signal, these two types of sensors can be fabricated at the end of a fiber and used as point sensors. Since the early 1990s, fiber Bragg grating ͑FBG͒ sensors have been intensively developed due to their many desirable advantages such as the small size, absolute measurement capability, immunity to electromagnetic interference, wavelength multiplexing, and distributed sensing possibilities. [1][2][3][4][5] Thus far, the FBG sensors' capability to measure physical quantities such as the temperature, strain, pressure, etc., has been studied extensively. [2][3][4][5][6][7][8] However, the use of FBG sensors for detection of environmental refractive index change has not been fully explored. Refractive index sensing is important for biological and chemical applications since a number of substances can be detected through measurements of the refractive index. [2][3][4][8][9][10][11][12][13] For normal FBGs, removal of the fiber cladding is required to increase the evanescent field interaction with the surrounding environment. This concept has been demonstrated using D-shaped fiber and sidepolished fiber. 9,11,12 In both cases, the strength and durability of the sensor were greatly reduced. Special fiber was also needed, which would raise the costs and limit the possible applications. Long-period fiber gratings have also been demonstrated to have high sensitivity to the refractive index of the ambient media, 2,3,13-15 however, their multiple resonance peaks and broad ͑typically tens of nanometers͒ transmission resonance features limit the measurement accuracy and their multiplexing capabilities. 9 In addition, the relatively long length of the grating limits their application as point sensor devices.In this letter, we first demonstrate a single etch-eroded FBG sensor using standard single-mode telecommunication fiber ͑Corning SMF-28͒. Fiber Fabry-Pérot interferometers ͑FFPIs͒ have also been widely used as sensors. 2,3,16,17 Compared to a single FBG, the FFPI sensors possess narrower resonance peaks and are more desirable for high accuracy wavelength measurement. 2,3,9,18 To that end we propose and demonstrate an etch-eroded FFPI sensor formed by two FBGs. The FFPI sensor is used to measure the refractive index of isopropyl alcohol ͑IPA͒ solutions of different concentrations, exhibiting the capabi...

The ability to manipulate and sort droplets is a fundamental issue in droplet-based microfluidics. Various lab-on-a-chip applications can only be realized if droplets are systematically categorized and sorted. These micron-sized droplets act as ideal reactors which compartmentalize different biological and chemical reagents. Array processing of these droplets hinges on the competence of the sorting and integration into the fluidic system. Recent technological advances only allow droplets to be actively sorted at the rate of kilohertz or less. In this review, we present state-of-the-art technologies which are implemented to efficiently sort droplets. We classify the concepts according to the type of energy implemented into the system. We also discuss various key issues and provide insights into various systems.

Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell wholetranscriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.genomics | lab on chip | embryonic stem cell

Abstract:We use the coupling matrix formalism to investigate continuouswave and pulse propagation through microring coupled-resonator optical waveguides (CROWs). The dispersion relation agrees with that derived using the tight-binding model in the limit of weak inter-resonator coupling. We obtain an analytical expression for pulse propagation through a semi-infinite CROW in the case of weak coupling which fully accounts for the nonlinear dispersive characteristics. We also show that intensity of a pulse in a CROW is enhanced by a factor inversely proportional to the inter-resonator coupling. In finite CROWs, anomalous dispersions allows for a pulse to propagate with a negative group velocity such that the output pulse appears to emerge before the input as in "superluminal" propagation. The matrix formalism is a powerful approach for microring CROWs since it can be applied to structures and geometries for which analyses with the commonly used tight-binding approach are not applicable.

Highlights d Comprehensive diagrams for comparing the features of the three systems d An open source versatile pipeline for all systems d Systematic comparison on sensitivity, precision, bias, and costs d Demonstration of Smart-seq2 protocols with inDrop platform