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Shahryari, A. ; Moya, N. ; Siehler, J. ; Wang, X. ; Burtscher, I. ; Lickert, H.

Increasing gene editing efficiency for CRISPR-Cas9 by small RNAs in pluripotent stem cells.

CRISPR J. 4, 491-501 (2021)

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Gene manipulations of human induced pluripotent stem cells (iPSCs) by CRISPR-Cas9 genome engineering are widely used for disease modeling and regenerative medicine applications. There are two competing pathways, non-homologous end joining (NHEJ) and homology directed repair (HDR) that correct the double-strand break generated by CRISPR-Cas9. Here, we improved gene editing efficiency of gene knock-in (KI) in iPSCs with minimum components by manipulating the Cas9 expression vector. Either we inserted short hairpin RNA expression cassettes to downregulate DNAPK and XRCC4, two main players of the NHEJ pathway, or we increased cell survival by inserting an anti-apoptotic expression cassette of miRNA-21 into the Cas9 vector. For an easy readout, the pluripotency gene SOX2 was targeted with a T2A-tdTomato reporter construct. In vitro downregulating DNAPK and XRCC4 increased the targeting efficiency of SOX2 KI by around twofold. Furthermore, co-expression of miRNA-21 and Cas9 improved the efficiency of SOX2 KI by around threefold. Altogether, our strategies provide a simple and valuable approach for efficient CRISPR-Cas9 gene editing in iPSCs.

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Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

Keywords Dna-pk; Genome; Repair; Identification; Inhibitor; Sox2

ISSN (print) / ISBN 2573-1599

e-ISSN 2573-1602

Journal The CRISPR Journal

Quellenangaben Volume: 4, Issue: 4, Pages: 491-501

Publisher Mary Ann Liebert

Publishing Place 140 Huguenot Street, 3rd Fl, New Rochelle, Ny 10801 Usa

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Institute of Diabetes and Regeneration Research (IDR)

Grants Deutsches Zentrum fur Diabetesforschung (DZD)
Helmholtz-Gemeinschaft (Helmholtz Portfolio Theme Metabolic Dysfunction and Common Disease)