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Crispy cas9

Thomas Gassler, Lina Heistinger, Diethard Mattanovich, Brigitte Gasser, Roland Prielhofer
State-of-the-art strain engineering techniques for the methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) include overexpression of endogenous and heterologous genes and deletion of host genes. For efficient gene deletion, methods such as the split-marker technique have been established. However, synthetic biology trends move toward building up large and complex reaction networks, which often require endogenous gene knockouts and simultaneous overexpression of individual genes or whole pathways...
2019: Methods in Molecular Biology
Emanuel Gonçalves, Fiona M Behan, Sandra Louzada, Damien Arnol, Euan A Stronach, Fengtang Yang, Kosuke Yusa, Oliver Stegle, Francesco Iorio, Mathew J Garnett
BACKGROUND: CRISPR-Cas9 genome editing is widely used to study gene function, from basic biology to biomedical research. Structural rearrangements are a ubiquitous feature of cancer cells and their impact on the functional consequences of CRISPR-Cas9 gene-editing has not yet been assessed. RESULTS: Utilizing CRISPR-Cas9 knockout screens for 250 cancer cell lines, we demonstrate that targeting structurally rearranged regions, in particular tandem or interspersed amplifications, is highly detrimental to cellular fitness in a gene-independent manner...
February 5, 2019: Genome Biology
Stephan Riesenberg, Tomislav Maricic
A now frequently used method to edit mammalian genomes uses the nucleases CRISPR/Cas9 and CRISPR/Cpf1 or the nickase CRISPR/Cas9n to introduce double-strand breaks which are then repaired by homology-directed repair using DNA donor molecules carrying desired mutations. Using a mixture of small molecules, the "CRISPY" mix, we achieve a 2.8- to 7.2-fold increase in precise genome editing with Cas9n, resulting in the introduction of the intended nucleotide substitutions in almost 50% of chromosomes or of gene encoding a blue fluorescent protein in 27% of cells, to our knowledge the highest editing efficiency in human induced pluripotent stem cells described to date...
June 4, 2018: Nature Communications
Gal Hyams, Shiran Abadi, Shlomtzion Lahav, Adi Avni, Eran Halperin, Eilon Shani, Itay Mayrose
The development of the CRISPR-Cas9 system in recent years has made eukaryotic genome editing, and specifically gene knockout for reverse genetics, a simple and effective task. The system is directed to a genomic target site by a programmed single-guide RNA (sgRNA) that base-pairs with it, subsequently leading to site-specific modifications. However, many gene families in eukaryotic genomes exhibit partially overlapping functions, and thus, the knockout of one gene might be concealed by the function of the other...
July 20, 2018: Journal of Molecular Biology
Kai Blin, Lasse Ebdrup Pedersen, Tilmann Weber, Sang Yup Lee
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...
June 2016: Synthetic and Systems Biotechnology
Carlotta Ronda, Lasse Ebdrup Pedersen, Henning Gram Hansen, Thomas Beuchert Kallehauge, Michael J Betenbaugh, Alex Toftgaard Nielsen, Helene Faustrup Kildegaard
Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47...
August 2014: Biotechnology and Bioengineering
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