Current Protocols in Molecular Biology

CRISPR-Cas9 has emerged as a powerful method for editing the genome in a wide variety of species, since it can generate a specific DNA break when targeted by the Cas9-bound guide RNA. In yeast, Cas9-targeted DNA breaks are used to promote homologous recombination with a mutagenic template DNA, in order to rapidly generate genome edits (e.g., DNA substitutions, insertions, or deletions) encoded in the template DNA. Since repeated Cas9-induced DNA breaks select against unedited cells, Cas9 can be used to generate marker-free genome edits...

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) system is being used successfully for efficient and targeted genome editing in various organisms, including the nematode Caenorhabditis elegans. Recent studies have developed a variety of CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: nonhomologous end joining and homologous recombination. Here, we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning (canonical marker-free and cassette selection methods), as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the germline...

Ribosome profiling quantifies the genome-wide ribosome occupancy of transcripts. With the integration of matched RNA sequencing data, the translation efficiency (TE) of genes can be calculated to reveal translational regulation. This layer of gene-expression regulation is otherwise difficult to assess on a global scale and generally not well understood in the context of human disease. Current statistical methods to calculate differences in TE have low accuracy, cannot accommodate complex experimental designs or confounding factors, and do not categorize genes into buffered, intensified, or exclusively translationally regulated genes...

Over the past century, formalin-fixed, paraffin-embedded (FFPE) tissue samples have represented the standard for basic histology and immunostaining. However, FFPE has several limitations and less stringent tissue preservation methods are required for the visualization of nucleic acids at high resolution, particularly those that are expressed at low levels. Here, we describe the FFPE properties that negatively impact RNA integrity, an alternative tissue preservation technique that prevents RNA loss, and the steps necessary to optimize slide preparation for single-molecule RNA fluorescent in situ hybridization (smRNA-FISH) and imaging by confocal microscopy...

Over the past decade, transcriptomic studies using next-generation sequencing (NGS)-based RNA sequencing (RNA-Seq) have greatly contributed to characterizing biochemical and physiological changes in cells and tissues across organisms and experimental conditions. Critical steps in RNA-Seq include the preparation of the sequencing library from extracted RNA. Currently, a large panoply of RNA-Seq kits are commercially available. In these kits, conversion of RNA into a sequencing library involves multiple steps, which are labor-intensive, and cost per sample for library preparation may limit routine use of RNA-Seq...

High-throughput screens in Drosophila melanogaster cell lines have led to discovery of conserved gene functions related to signal transduction, host-pathogen interactions, ion transport, and more. CRISPR/Cas9 technology has opened the door to new types of large-scale cell-based screens. Whereas array-format screens require liquid handling automation and assay miniaturization, pooled-format screens, in which reagents are introduced at random and in bulk, can be done in a standard lab setting. We provide a detailed protocol for conducting and evaluating genome-wide CRISPR single guide RNA (sgRNA) pooled screens in Drosophila S2R+ cultured cells...

Tagging proteins with fluorescent reporters such as green fluorescent protein (GFP) is a powerful method to determine protein localization, especially when proteins are tagged in the endogenous context to preserve native genomic regulation. However, insertion of fluorescent reporters into the genomes of mammalian cells has required the construction of plasmids containing selection markers and/or extended sequences homologous to the site of insertion (homology arms). Here we describe a streamlined protocol that eliminates all cloning steps by taking advantage of the high propensity of linear DNAs to engage in homology-directed repair of DNA breaks induced by the Cas9 RNA-guided endonuclease...

The budding yeast, Saccharomyces cerevisiae, has been widely used for genetic studies of fundamental cellular functions. The isolation and analysis of yeast mutants is a commonly used and powerful technique to identify the genes that are involved in a process of interest. Furthermore, natural genetic variation among wild yeast strains has been studied for analysis of polygenic traits by quantitative trait loci mapping. Whole-genome sequencing, often combined with bulk segregant analysis, is a powerful technique that helps determine the identity of mutations causing a phenotype...

The auxin-inducible degron (AID) is a powerful tool that is used for depletion of proteins to study their function in vivo. This method can conditionally induce the degradation of any protein by the proteasome simply by the addition of the plant hormone auxin. This approach is particularly valuable to study the function of essential proteins. The protocols provided here describe the steps to construct the necessary strains and to optimize auxin-inducible depletion in Saccharomyces cerevisiae. © 2019 by John Wiley & Sons, Inc...

The DMS region extraction and deep sequencing (DREADS) procedure was designed to probe RNA structure in vivo and to link this structural information to specific 3' isoforms. Growing cells are treated with the alkylating agent dimethyl sulfate (DMS), which enters easily into cells and modifies RNA molecules at solvent-exposed A and C residues. RNA is isolated, and sequencing libraries are constructed in a manner that preserves the identities of individual mRNA isoforms arising from alternative cleavage/polyadenylation sites...

CRISPR/Cas9 technology enables the rapid generation of loss-of-function mutations in a targeted gene in mammalian cells. A single cell harboring those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of biological reagents. However, the successful derivation of knockout clones can be technically challenging and may be complicated by multiple factors, including incomplete target ablation and interclonal heterogeneity...

The identification of transcriptional enhancers and the quantitative assessment of enhancer activities is essential to understanding how regulatory information for gene expression is encoded in animal and human genomes. Further, it is key to understanding how sequence variants affect enhancer function. STARR-seq enables the direct and quantitative assessment of enhancer activity for millions of candidate sequences of arbitrary length and origin in parallel, allowing the screening of entire genomes and the establishment of genome-wide enhancer activity maps...

Here we describe CLIP-READS, a technique that combines elements of crosslinking and immunoprecipitation (CLIP) and 3' region extraction and deep sequencing (READS), to provide a genome-wide map of mRNA 3' isoform binding by a given messenger ribonucleoprotein (mRNP). In CLIP-READS, cells are grown to logarithmic phase and are irradiated with UV light (254 nm) to form RNA-protein adducts. The protein-mRNA complexes are immunoprecipitated from cell extracts with an antibody specific to the protein of interest, after which the protein component is digested away with Pronase...

MicroRNAs (miRNAs) are key regulators of cell and tissue development. However, spatial resolution of miRNA heterogeneity and accumulation patterns in vivo remains uncharted. Next-generation sequencing methods assay miRNA abundance in tissues, yet these analyses do not provide spatial resolution. A method to assay miRNA expression at single-cell resolution in vivo should clarify the cell-autonomous functions of miRNAs, their roles in influencing the cellular microenvironment, and their perdurance and turnover rate...

Cell death involves the release of short DNA fragments into blood, termed circulating cell-free DNA (cfDNA). Sequencing of cfDNA in the plasma has recently emerged as a liquid biopsy for detecting fetal chromosomal aberrations, tumor DNA, and graft rejection. However, in cases where cfDNA is derived from tissues with a normal genome, its primary sequence is not informative regarding the tissue of origin. We developed a method of determining the tissue origins of cfDNA, allowing inference of tissue-specific cell death, based on tissue-specific methylation patterns...

An understanding of the dynamic structural properties of chromatin requires techniques that allow the profiling of regions of both open and closed chromatin as well as the assessment of nucleosome occupancy. The recently developed MNase accessibility (MACC) technique allows for the simultaneous measurement of chromatin opening and compaction, as well as nucleosome occupancy, on a genome-wide scale in a single assay. This article presents a low-input MACC procedure that considerably extends the utility of the original MACC assay...

Genetic tools for specific perturbation of endogenous gene expression are highly desirable for interrogation of plant gene functions and improvement of crop traits. Synthetic transcriptional activators derived from the CRISPR/Cas9 system are emerging as powerful new tools for activating the endogenous expression of genes of interest in plants. These synthetic constructs, generated by tethering transcriptional activation domains to a nuclease-dead Cas9 (dCas9), can be directed to the promoters of endogenous target genes by single guide RNAs (sgRNAs) to activate transcription...

Quantitative analysis of single-cell RNA sequencing (RNA-seq) is crucial for discovering the heterogeneity of cell populations and understanding the molecular mechanisms in different cells. In this unit we present a bioinformatics workflow for analyzing single-cell RNA-seq data with a few current publicly available computational tools. This workflow is focused on the interpretation of the heterogeneity from single-cell transcriptomes as well as the identification of cell clusters and genes that are differentially expressed between clusters...

Determining the genomic location of DNA-binding proteins is essential to understanding their function. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a powerful method for mapping protein-DNA interactions at high resolution. In CUT&RUN, a recombinant protein A-microccocal nuclease (pA-MN) fusion is recruited by an antibody targeting the chromatin protein of interest; this can be done with either uncrosslinked or formaldehyde-crosslinked cells. DNA fragments near sites of antibody binding are released from the insoluble bulk chromatin through endonucleolytic cleavage and used to build barcoded DNA-sequencing libraries that can be sequenced in pools of at least 30...

The most prevalent modified base in mRNA, N6 -methyladenosine (m6 A), is found in several thousand transcripts, typically near the stop codon, although it can occur anywhere in the mRNA. In addition, the highly similar nucleotide N6 ,2'-O-dimethyladenosine (m6 Am), which is difficult to distinguish from m6 A, occurs as the first transcribed nucleotide of certain transcripts. Both the m6 A and m6 Am modifications have been implicated in numerous biological processes, and their precise mapping is crucial to understanding their functions...