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Genes 2018, 9(6), 296; https://doi.org/10.3390/genes9060296

Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene

1
Institute for Molecular and Cell Physiology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
2
Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Hoeltystrasse 10, Mariensee, 31535 Neustadt, Germany
Deceased.
*
Author to whom correspondence should be addressed.
Received: 23 April 2018 / Revised: 30 May 2018 / Accepted: 8 June 2018 / Published: 13 June 2018
(This article belongs to the Section Animal Genetics and Genomics)
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Abstract

During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the MYH7-gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene (GMNN). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9-GMNN and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9-GMNN provides a promising way to enrich specific knock-in in porcine fibroblast cultures for somatic cloning approaches. View Full-Text
Keywords: genome editing; pig; CRISPR/Cas9; geminin; myosin; porcine fetal fibroblasts; SCR7 genome editing; pig; CRISPR/Cas9; geminin; myosin; porcine fetal fibroblasts; SCR7
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Gerlach, M.; Kraft, T.; Brenner, B.; Petersen, B.; Niemann, H.; Montag, J. Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene. Genes 2018, 9, 296.

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