Error-corrected next generation sequencing - Promises and challenges for genotoxicity and cancer risk assessment.

Error-corrected Next Generation Sequencing (ecNGS) is rapidly emerging as a valuable, highly sensitive and accurate method for detecting and characterizing mutations in any cell type, tissue or organism from which DNA can be isolated. Recent mutagenicity and carcinogenicity studies have used ecNGS to quantify drug-/chemical-induced mutations and mutational spectra associated with cancer risk. ecNGS has potential applications in genotoxicity assessment as a new readout for traditional models, for mutagenesis studies in 3D organotypic cultures, and for detecting off-target effects of gene editing tools. Additionally, early data suggest that ecNGS can measure clonal expansion of mutations as a mechanism-agnostic early marker of carcinogenic potential and can evaluate mutational load directly in human biomonitoring studies. In this review, we discuss promising applications, challenges, limitations, and key data initiatives needed to enable regulatory testing and adoption of ecNGS - including for advancing safety assessment, augmenting weight-of-evidence for mutagenicity and carcinogenicity mechanisms, identifying early biomarkers of cancer risk, and managing human health risk from chemical exposures.

Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations.

Base editors are recent multiplex gene editing tools derived from the Cas9 nuclease of Streptomyces pyogenes. They can target and modify a single nucleotide in the genome without inducing double-strand breaks (DSB) of the DNA helix. As such, they hold great potential for the engineering of microbes that lack effective DSB repair pathways such as homologous recombination (HR) or non-homologous end-joining (NHEJ). However, few applications of base editors have been reported in prokaryotes to date, and their advantages and drawbacks have not been systematically reported. Here, we used the base editors Target-AID and Target-AID-NG to introduce nonsense mutations into four different coding sequences of the industrially relevant Gram-positive bacterium Clostridium autoethanogenum. While up to two loci could be edited simultaneously using a variety of multiplexing strategies, most colonies exhibited mixed genotypes and most available protospacers led to undesired mutations within the targeted editing window. Additionally, fifteen off-target mutations were detected by sequencing the genome of the resulting strain, among them seven single-nucleotide polymorphisms (SNP) in or near loci bearing some similarity with the targeted protospacers, one 15 nt duplication, and one 12 kb deletion which removed uracil DNA glycosylase (UDG), a key DNA repair enzyme thought to be an obstacle to base editing mutagenesis. A strategy to process prokaryotic single-guide RNA arrays by exploiting tRNA maturation mechanisms is also illustrated.

Tips, Tricks, and Potential Pitfalls of CRISPR Genome Editing in Saccharomyces cerevisiae.

The versatility of clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) genome editing makes it a popular tool for many research and biotechnology applications. Recent advancements in genome editing in eukaryotic organisms, like fungi, allow for precise manipulation of genetic information and fine-tuned control of gene expression. Here, we provide an overview of CRISPR genome editing technologies in yeast, with a particular focus on Saccharomyces cerevisiae. We describe the tools and methods that have been previously developed for genome editing in Saccharomyces cerevisiae and discuss tips and experimental tricks for promoting efficient, marker-free genome editing in this model organism. These include sgRNA design and expression, multiplexing genome editing, optimizing Cas9 expression, allele-specific editing in diploid cells, and understanding the impact of chromatin on genome editing. Finally, we summarize recent studies describing the potential pitfalls of using CRISPR genome targeting in yeast, including the induction of background mutations.

Genetically-modified cell lines: categorisation and considerations for characterisation.

Stem Cell Research is pleased to introduce into its publication portfolio a new article type: a template-driven short report on the generation of a novel Genetically Modified Cell Line. This resource type is typically derived from human pluripotent stem cell lines via the introduction of nucleases and/or foreign genetic material leading to stable genomic alterations, maintained in a single cell-derived clonal cell line. Interest in, and demand for, genetically modified cell lines has grown exponentially in the last few years. This overview provides a brief introduction to this incredibly versatile lab resource and marks the beginning of a new and exciting addition to the publication portfolio of Stem Cell Research. A dramatic increase in the accessibility of the human genome in the last decade has given a long-anticipated boost to advanced biomedical studies in human in vitro systems. Pluripotent stem cells represent a particularly attractive gateway into this line of experimentation due to their unique suitability for the isolation of clonal genetically modified cell lines (GMCLs), and the ability to be differentiated into essentially any cell type upon the lines' virtually limitless expansion.

Programmable Molecular Scissors: Applications of a New Tool for Genome Editing in Biotech.

Targeted genome editing is an advanced technique that enables precise modification of the nucleic acid sequences in a genome. Genome editing is typically performed using tools, such as molecular scissors, to cut a defined location in a specific gene. Genome editing has impacted various fields of biotechnology, such as agriculture; biopharmaceutical production; studies on the structure, regulation, and function of the genome; and the creation of transgenic organisms and cell lines. Although genome editing is used frequently, it has several limitations. Here, we provide an overview of well-studied genome-editing nucleases, including single-stranded oligodeoxynucleotides (ssODNs), transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and CRISPR-Cas9 RNA-guided nucleases (CRISPR-Cas9). To this end, we describe the progress toward editable nuclease-based therapies and discuss the minimization of off-target mutagenesis. Future prospects of this challenging scientific field are also discussed.