CRISPy-web:An online resource to design sgRNAs for CRISPR applications

CRISPR/Cas9-based genome editing has been one of the major achievements of molecular biology, allowing the targeted engineering of a wide range of genomes. The system originally evolved in prokaryotes as an adaptive immune system against bacteriophage infections. It now sees widespread application in genome engineering workflows, especially using the Streptococcus pyogenes endonuclease Cas9. To utilize Cas9, so-called single guide RNAs (sgRNAs) need to be designed for each target gene. While there are many tools available to design sgRNAs for the popular model organisms, only few tools that allow designing sgRNAs for non-model organisms exist. Here, we present CRISPy-web (http://crispy.secondarymetabolites.org/), an easy to use web tool based on CRISPy to design sgRNAs for any user-provided microbial genome. CRISPy-web allows researchers to interactively select a region of their genome of interest to scan for possible sgRNAs. After checks for potential off-target matches, the resulting sgRNA sequences are displayed graphically and can be exported to text files. All steps and information are accessible from a web browser without the requirement to install and use command line scripts

CRMAGE: CRISPR Optimized MAGE Recombineering

A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli. Using CRMAGE, the recombineering efficiency was between 96.5% and 99.7% for gene recoding of three genomic targets, compared to between 0.68% and 5.4% using traditional recombineering. For modulation of protein synthesis (small insertion/RBS substitution) the efficiency was increased from 6% to 70%. CRMAGE can be multiplexed and enables introduction of at least two mutations in a single round of recombineering with similar efficiencies. PAM-independent loci were targeted using degenerate codons, thereby making it possible to modify any site in the genome. CRMAGE is based on two plasmids that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red oligos and the gRNAs. The CRMAGE platform enables highly efficient and fast genome editing and may open up promising prospective for automation of genome-scale engineering

CRISPR activation screening of dormant genes to improve secretory capacity in CHO cells

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CRISPR-CAS9 knockout library for CHO

Traditionally, screening of large CHO cell population have been utilized to identify clones with desired phenotypic properties such as product quality, e.g. specific glyco forms, and population characteristics, e.g. ability to grow in high cell densities. This has largely depended on the genomic variety naturally present in a large cell population or occasionally utilizing random mutagenesis to increase this variety. The ability to precisely create genomic variety in mammalian cells have improved dramatically over the past decade and in the past few years the price has dropped substantially due to the CRISPR/Cas9 technology. E.g. knocking out a gene using CRISPR/Cas9 is a simple, fast and cheap process (1). However, rational identification of which genes to target can be quite difficult and the cellular processes underlying many desired traits are simply unknown or only poorly/partially understood. To both improve our understanding of phenotypes of interest and identify targets to modify, we have created a lentiviral guideRNA library against CHO genes. Using this guideRNA library we subject cells to various phenotype selection assays, harvest genomic DNA from the selected cells and perform targeted next generation sequencing to identify the guideRNA sequences which led to the improved phenotype. As an example: Using a toxic fucose binding lectin, one can potentially identify all genes required for fucosylation in one experiment, simply by identifying the guideRNA present in the surviving population