Genomic integration of unclonable gene expression cassettes in Saccharomyces cerevisiae using rapid cloning-free workflows.

Most DNA assembly methods require bacterial amplification steps, which restrict its application to genes that can be cloned in the bacterial host without significant toxic effects. However, genes that cannot be cloned in bacteria do not necessarily exert toxic effects on the final host. In order to tackle this issue, we adapted two DNA assembly workflows for rapid, cloning-free construction and genomic integration of expression cassettes in Saccharomyces cerevisiae. One method is based on a modified Gibson assembly, while the other relies on a direct assembly and integration of linear PCR products by yeast homologous recombination. The methods require few simple experimental steps, and their performance was evaluated for the assembly and integration of unclonable zeaxanthin epoxidase expression cassettes in yeast. Results showed that up to 95% integration efficiency can be reached with minimal experimental effort. The presented workflows can be employed as rapid gene integration tools for yeast, especially tailored for integrating unclonable genes.

MELC Genomics: A Framework for De Novo Genome Assembly.

The development of next-generation sequencing platforms increased substantially the capacity of data generation. In addition, in the past years, the costs for whole genome sequencing have been reduced that made it easier to access this technology. As a result, the storage and analysis of the data generated became a challenge, ushering in the development of bioinformatic tools, such as programs and programming languages, able to store, process, and analyze this huge amount of information. In this article, we present MELC genomics, a framework for genome assembly in a simple and fast workflow.

CRISPR-based tools for plant genome engineering.

Molecular tools adapted from bacterial CRISPR (clustered regulatory interspaced short palindromic repeat) adaptive immune systems have been demonstrated in an increasingly wide range of plant species. They have been applied for the induction of targeted mutations in one or more genes as well as for directing the integration of new DNA to specific genomic loci. The construction of molecular tools for multiplexed CRISPR-mediated editing in plants has been facilitated by cloning techniques that allow multiple sequences to be assembled together in a single cloning reaction. Modifications of the canonical Cas9 protein from Streptococcus pyogenes and the use of nucleases from other bacteria have increased the diversity of genomic sequences that can be targeted and allow the delivery of protein cargos such as transcriptional activators and repressors. Furthermore, the direct delivery of protein-RNA complexes to plant cells and tissues has enabled the production of engineered plants without the delivery or genomic integration of foreign DNA. Here, we review toolkits derived from bacterial CRISPR systems for targeted mutagenesis, gene delivery and modulation of gene expression in plants, focusing on their composition and the strategies employed to reprogramme them for the recognition of specific genomic targets.