Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells

Stephan Riesenberg () and Tomislav Maricic
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Stephan Riesenberg: Max-Planck-Institute for Evolutionary Anthropology
Tomislav Maricic: Max-Planck-Institute for Evolutionary Anthropology

Nature Communications, 2018, vol. 9, issue 1, 1-9

Abstract: Abstract 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. Furthermore, the CRISPY mix improves precise genome editing with Cpf1 2.3- to 4.0-fold, allowing almost 20% of chromosomes to be edited. The components of the CRISPY mix do not always increase the editing efficiency in the immortalized or primary cell lines tested, suggesting that employed repair pathways are cell-type specific.

Date: 2018
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DOI: 10.1038/s41467-018-04609-7

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