Orthogonal Genetic Regulation in Human Cells Using Chemically Induced CRISPR/Cas9 Activators

The concerted action of multiple genes in a time-dependent manner controls complex cellular phenotypes, yet the temporal regulation of gene expressions is restricted on a single-gene level, which limits our ability to control higher-order gene networks and understand the consequences of multiplex genetic perturbations. Here we developed a system for temporal regulation of multiple genes. This system combines the simplicity of CRISPR/Cas9 activators for orthogonal targeting of multiple genes and the orthogonality of chemically induced dimerizing (CID) proteins for temporal control of CRISPR/Cas9 activator function. In human cells, these transcription activators exerted simultaneous activation of multiple genes and orthogonal regulation of different genes in a ligand-dependent manner with minimal background. We envision that our system will enable the perturbation of higher-order gene networks with high temporal resolution and accelerate our understanding of gene–gene interactions in a complex biological setting

Interaction of copper (II) complexes by bovine serum albumin: spectroscopic and calorimetric insights

<p>Serum albumins being the most abundant proteins in the blood and cerebrospinal fluid are significant carriers of essential transition metal ions in the human body. Studies of copper (II) complexes have gained attention because of their potential applications in synthetic, biological, and industrial processes. Study of binding interactions of such bioinorganic complexes with serum albumins improves our understanding of biomolecular recognition process essential for rational drug design. In the present investigation, we have applied quantitative approach to explore interactions of novel synthesized copper (II) complexes <i>viz</i>. [Cu(L¹)(L²)ClO₄] (complex I), [Cu(L²)(L³)]ClO₄] (complex II) and [Cu(L⁴)₂(H₂O)₂] (complex III) with bovine serum albumin (BSA) to evaluate their binding characteristics, site and mode of interaction. The fluorescence quenching of BSA initiated by complexation has been observed to be static in nature. The binding interactions are endothermic driven by entropic factors as confirmed by high sensitivity isothermal titration calorimetry. Changes in secondary and tertiary structure of protein have been studied by circular dichroism and significant reduction in α-helical content of BSA was observed upon binding. Site marking experiments with warfarin and ibuprofen indicated that copper complexes bind at site II of the protein.</p

A Scalable Epitope Tagging Approach for High Throughput ChIP-Seq Analysis

Eukaryotic transcriptional factors (TFs) typically recognize short genomic sequences alone or together with other proteins to modulate gene expression. Mapping of TF-DNA interactions in the genome is crucial for understanding the gene regulatory programs in cells. While chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is commonly used for this purpose, its application is severely limited by the availability of suitable antibodies for TFs. To overcome this limitation, we developed an efficient and scalable strategy named cmChIP-Seq that combines the clustered regularly interspaced short palindromic repeats (CRISPR) technology with microhomology mediated end joining (MMEJ) to genetically engineer a TF with an epitope tag. We demonstrated the utility of this tool by applying it to four TFs in a human colorectal cancer cell line. The highly scalable procedure makes this strategy ideal for ChIP-Seq analysis of TFs in diverse species and cell types

ChimericSeq: An open-source, user-friendly interface for analyzing NGS data to identify and characterize viral-host chimeric sequences

<div><p>Identification of viral integration sites has been important in understanding the pathogenesis and progression of diseases associated with particular viral infections. The advent of next-generation sequencing (NGS) has enabled researchers to understand the impact that viral integration has on the host, such as tumorigenesis. Current computational methods to analyze NGS data of virus-host junction sites have been limited in terms of their accessibility to a broad user base. In this study, we developed a software application (named ChimericSeq), that is the first program of its kind to offer a graphical user interface, compatibility with both Windows and Mac operating systems, and optimized for effectively identifying and annotating virus-host chimeric reads within NGS data. In addition, ChimericSeq’s pipeline implements custom filtering to remove artifacts and detect reads with quantitative analytical reporting to provide functional significance to discovered integration sites. The improved accessibility of ChimericSeq through a GUI interface in both Windows and Mac has potential to expand NGS analytical support to a broader spectrum of the scientific community.</p></div

Evaluation of integration sites from NGS data of HBV-infected patients.

<p>Evaluation of integration sites from NGS data of HBV-infected patients.</p

Schematic overview of the ChimericSeq workflow.

<p>Input NGS reads are manually loaded through a graphical interface, followed by user-determined 5’ and 3’ end trimming. Host and viral genomes and indices must be identified, if not otherwise already loaded. Next, the identification phase aligns each read to the specified viral genome, extracts these aligned reads, and then aligns the reads to the host genome. The identification phase is further broken down to describe potential scenarios, where 1) the read has no alignment to the viral genome, and is thus discarded, as indicated by the “X”, 2) the read has alignment to the viral genome, however the unmapped region’s length is lower than the threshold set by the program (or user), and is thus discarded, and 3) the read has alignment to the viral genome and has sufficient unmapped overhang for alignment to the host genome, and is extracted (as indicated by the checkmark). The extracted reads are then subjected to Bowtie2 alignment to the host genome, following similar scenarios as depicted. The identified chimeric reads are then passed to the post processing phase, which includes steps to filter out artifacts and annotate integration sites with functional information such as gene breakpoint location. Finally, reads are presented through the program interface and saved to accessible output files.</p

Differential PD-1 expression on A2-BMLF-1 and A2-BRLF-1 CD8+ T cells at AIM and convalescence.

<p>PD-1 expression on A2-BMLF-1 and A2-BRLF-1 CD8+ T cells during AIM (upper panels) and convalescence (lower panels). Representative dot plots demonstrate differences in PD-1 percent positivity, and histograms showing differences in distribution and PD-1 MFI of these EBV-specific CD8+ T cells. Histograms overlay the two subsets on a background of the total CD8+ T cell population.</p

Percent detection of HBV integration sites with defined lengths of viral DNA insertion.

<p>Percent detection of HBV integration sites with defined lengths of viral DNA insertion.</p

Comparison of required third-party dependencies.

<p>Comparison of required third-party dependencies.</p

High level PD-1 expression on CD8+ T cells during AIM and convalescence.

<p><b>A</b>. Representative CD8+ T cell plots of a sample obtained during AIM demonstrating the gating strategy to define PD-1 high, dim and negative populations. <b>B</b>. Percentages of PD-1 high, dim, or negative CD8+ T cells in AIM and convalescence. <b>C</b>. Percentages of PD-1 high, dim, or negative A2-BMLF-1 and A2-BRLF-1 CD8+ T cells in AIM and convalescence. Lines define paired results for each individual. Brackets indicate that differences between selected comparisons are significant (Wilcoxon signed rank test; *p<0.05, ** p<0.01, *** p<0.001).</p