Volume 16 Issue 1, January 2021

Off-target effects of programmable nucleases remain a critical issue for therapeutic applications of genome editing. This review compares experimental and computational tools for off-target analysis and provides recommendations for better assessments of off-target effects.

This protocol extension describes an improved method for global profiling of poly(A) RNA-binding proteins (RBPs) and quantitative analysis of RBP dynamics in response to biological and pharmacological cues that uses UV crosslinking, capture with LNA-modified oligo-dT probes, and proteomics.

Chemicals are used to induce sporadic and inflammation-associated colon tumor growth in mouse models. When combined with p53 deficiency, tumor development is promoted, including the growth of invasive cancers and lymph node metastasis.

This protocol provides a detailed guide for the design and assembly of membrane-spanning DNA nanopores and includes assays for characterizing channel function.

Here, we present a protocol for producing exosomes loaded with ultrathin Pd nanosheets for targeted bio-orthogonal catalysis. Pd precursors are loaded into exosomes by diffusion and reduced into metallic nanosheets by using gas-phase CO.

This protocol describes how to convert a commercial confocal spinning-disk microscope into an image scanning microscope (ISM), allowing fast imaging of fixed and live samples with increases of up to twofold in resolution and fourfold in contrast.

Culture conditions are described for long-term expansion of human fetal hepatocytes as 3D organoids. Gene knockin and knockout approaches are also described for organoids derived from human fetal hepatocytes and human adult liver ductal cells.

This protocol provides ImageJ-based workflows for the analysis of images obtained from colorimetric assays. New users can take advantage of a basic workflow; more experienced users can benefit from more advanced analysis procedures.

This protocol describes how to perform antigen retrieval and tissue clearing for volumetric imaging of whole organs, organoids and small organisms by using fast light-microscopic analysis of antibody-stained whole organs.

This protocol describes the construction, calibration and use of the Drosophila traumatic brain injury (dTBI) device to induce different degrees of TBI in flies. Two versions of the device are described, for low- and high-throughput applications.

Phosphatidylethanol (PEth) is a metabolite of ethanol that can be used as a stable, direct marker of alcohol usage. This protocol describes how to determine the concentration of PEth in dry blood spots via manual or automated extraction followed by LC-MS/MS.

Human pluripotent stem cells are seeded in a microfluidic device to establish a model that resembles the progressive development of the early post-implantation human embryo.

Delivery of siRNAs to immune cells is challenging because of these cells’ sensitivity to standard transfection reagents. This protocol describes how to synthesize an amphiphilic dendrimer and apply it to transfect siRNAs in a variety of primary immune cells.

This protocol describes the fabrication of 3D collagen scaffolds used to culture human colonic crypts derived from primary intestinal epithelial cells.

We report a new protocol for tracking endocytosis and cellular distribution of spherical nucleic acid nanoparticles of different clustering states by correlative imaging with dark-field microscopy, fluorescence microscopy and scanning electron microscopy.

This article includes two protocols for the construction of responsive and activable nanomedicines that regulate the tumor microenvironment.

This protocol provides a whole-genome sequencing (WGS) method for evaluating the specificity of base editors in rice.

This is a protocol for the extraction of microgram quantities of high-molecular-weight DNA from human stool samples that are suitable for downstream long-read sequencing applications.

This protocol describes a set of tools and procedures for (1) intravital labeling for the identification of mural cells, (2) in vivo calcium imaging of pericytes and vSMCs, and (3) single- and two-photon optogenetics to study blood flow control.

Mechanical sample motion (drift) is a major factor that limits fluorophore localization precision during single-molecule localization microscopy. This protocol describes how to implement 3D active stabilization onto custom and standard microscopes.

This protocol describes a strategy for revealing the initial interactions between host proteins and incoming viral RNAs that relies on solid-phase purification of crosslinked RNA–protein complexes followed by mass spectrometry.

This protocol describes how to implement high-speed single-point edge-excitation sub-diffraction (SPEED) microscopy in combination with 2D-to-3D transformation to obtain 3D sub-diffraction-limited information about rotational symmetric structures.