Increasing Gene Editing Efficiency for CRISPR-Cas9 by Small RNAs in Pluripotent Stem Cells

Alireza Shahryari, Noel Moya, Johanna Siehler, Xianming Wang, Ingo Burtscher, Heiko Lickert

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)491-501
Number of pages11
JournalCRISPR Journal
Volume4
Issue number4
DOIs
StatePublished - Aug 2021
Externally publishedYes

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