Quantitative proteomics of cancer cell lines

Nature Cell Biology

he transformation from DNA to protein is a complex, multi-stage process that revolves around RNA metabolism. After transcription, RNA molecules proceed to splicing, localization, translation and degradation. These steps are highly coordinated and tightly regulated in both spatial and temporal domains. Traditional biochemistry and genetic tools have elucidated some of the what and the how, such as the identities and functions of proteins and non-coding RNAs (ncRNAs) involved in each step of RNA processing. To delve deeper into the when and where, methods to visualize RNA within cells are required. Towards this goal, in the past four decades groups have developed and advanced RNA imaging tools for both fixed and live cells (Fig. 1 and Table 1). These RNA imaging tools take advantage of recent and rapid innovation in fluorescent microscopy, image processing, DNA chemistry and next-generation sequencing to achieve multiple milestones, including single-molecule sensitivity, super-resolution, multiplexing and live-cell RNA tracking. In this Review we discuss the developments in RNA imaging and the RNA biology they have and are poised to unravel. RNA imaging technologies RNA imaging technologies have been evolving rapidly for both fixed and live cells. In fixed cells, current methods have achieved substantial throughput and are capable of detecting localization and quantifying the expression level of the whole transcriptome. In live cells, throughput is limited to a single gene per colour; however, the temporal resolution of live-cell RNA imaging has significantly advanced our understanding of the dynamics of RNA processing.

Annual review of …, 2009

The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding.

Science (New York, N.Y.), 2015

The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.

ACS Central Science

The recently discovered CRISPR-Cas gene editing system and its derivatives have found numerous applications in fundamental biology research and pharmaceutical sciences. The need for precise external control over the gene editing and regulatory events has driven the development of inducible CRISPR-Cas systems. While most of the light-controllable CRISPR-Cas systems are based on protein engineering, we developed an alternative synthetic approach based on modification of crRNA/ tracrRNA duplex (guide RNA or gRNA) with photocaging groups, preventing the gRNA from recognizing its genome target sequence until its deprotection is induced within seconds of illumination. This approach relies on a straightforward solid-phase synthesis of the photocaged gRNAs, with simpler purification and characterization processes in comparison to engineering a lightresponsive protein. We have demonstrated the feasibility of photocaging of gRNAs and light-mediated DNA cleavage upon brief exposure to light in vitro. We have achieved light-mediated spatiotemporally resolved gene editing as well as gene activation in cells, whereas photocaged gRNAs showed virtually no detectable gene editing or activation in the absence of light irradiation. Finally, we have applied this system to spatiotemporally control gene editing in zebrafish embryos in vivo, enabling the use of this strategy for developmental biology and tissue engineering applications.

The ability to remotely trigger CRISPR/Cas9 activity would enable new strategies to study cellular events with greater precision and complexity. In this work, we have developed a method to photocage the activity of the guide RNA called " CRISPR-plus " (CRISPR-precise light-mediated unveiling of sgRNAs). The photoactivation capability of our CRISPR-plus method is compatible with the simultaneous targeting of multiple DNA sequences and supports numerous modifications that can enable guide RNA labeling for use in imaging and mechanistic investigations.

Cell reports, 2017

CRISPR-Cas systems defend bacteria and archaea against infection by bacteriophage and other threats. The central component of these systems are surveillance complexes that use guide RNAs to bind specific regions of foreign nucleic acids, marking them for destruction. Surveillance complexes must locate targets rapidly to ensure timely immune response, but the mechanism of this search process remains unclear. Here, we used single-molecule FRET to visualize how the type I-E surveillance complex Cascade searches DNA in real time. Cascade rapidly and randomly samples DNA through nonspecific electrostatic contacts, pausing at short PAM recognition sites that may be adjacent to the target. We identify Cascade motifs that are essential for either nonspecific sampling or positioning and readout of the PAM. Our findings provide a comprehensive structural and kinetic model for the Cascade target-search mechanism, revealing how CRISPR surveillance complexes can rapidly search large amounts of g...

We developed a new method for conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos via photochemically activated, caged guide RNAs. Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5’-protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:target dsDNA hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off to on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. caged gRNAs are novel tools for conditional control of gene editing thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.

Biochemistry

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system is an adaptive immune system of bacteria that has furnished several RNA-guided DNA endonucleases (e.g., Cas9) that are revolutionizing the field of genome engineering. Cas9 is being used to effect genomic alterations as well as in gene drives, where a particular trait may be propagated through a targeted species population over several generations. The ease of targeting catalytically impaired Cas9 to any genomic loci has led to development of technologies for base editing, chromatin imaging and modeling, epigenetic editing, and gene regulation. Unsurprisingly, Cas9 is being developed for numerous applications in biotechnology and biomedical research and as a gene therapy agent for multiple pathologies. There is a need for precise control of Cas9 activity over several dimensions, including those of dose, time, and space in these applications. Such precision controls, which are required of therapeutic agents, are particularly important for Cas9 as off-target effects, chromosomal translocations, immunogenic response, genotoxicity, and embryonic mosaicism are observed at elevated levels and with prolonged activity of Cas9. Here, we provide a perspective on advances in the precision control of Cas9 over aforementioned dimensions using external stimuli (e.g., small molecules or light) for controlled activation, inhibition, or degradation of Cas9.

JESHO, 2024

This article is the first in-depth study of an Ilkhanid private document from the Mausoleum of Shaykh Ṣafī in Ardabil (North-Western Iran). It contains the critical edition, translation and commentary of an original deed recording the sale of half a village near Miyāna, in the south of the Iranian province of Azarbayjan, in 666/1267. The document is remarkable for its length, its highly literary wording, some formal oddities and also the identity of the parties. It shows that the seller, Bint Toghrïl, daughter of the last Saljuq sultan of Iran, whose life is documented by chronicles, managed not only to survive the Mongol conquests but also somehow to retain her economic base for several decades. The identity of the buyer and the qadi who legalized the act illustrates the strong presence of powerful Shafiʿi families in Mongol Iran (here the Qazwīnīs and Mākī Qazwīnīs). We compare this deed of sale with other unpublished items of the Ardabil corpus as well as similar documents from better-known medieval archival funds from the Near East.