Precise CCM1 gene correction and inactivation in patient-derived endothelial cells: Modeling Knudson’s two-hit hypothesis in vitro.

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Precise CCM1 gene correction and inactivation in patient-derived endothelial cells: Modeling Knudson’s two-hit hypothesis in vitro.

Mol Genet Genomic Med. 2019 07;7(7):e00755

Authors: Spiegler S, Rath M, Much CD, Sendtner BS, Felbor U

Abstract
BACKGROUND: The CRISPR/Cas9 system has opened new perspectives to study the molecular basis of cerebral cavernous malformations (CCMs) in personalized disease models. However, precise genome editing in endothelial and other hard-to-transfect cells remains challenging.
METHODS: In a proof-of-principle study, we first isolated blood outgrowth endothelial cells (BOECs) from a CCM1 mutation carrier with multiple CCMs. In a CRISPR/Cas9 gene correction approach, a high-fidelity Cas9 variant was then transfected into patient-derived BOECs using a ribonucleoprotein complex and a single-strand DNA oligonucleotide. In addition, patient-specific CCM1 knockout clones were expanded after CRISPR/Cas9 gene inactivation.
RESULTS: Deep sequencing demonstrated correction of the mutant allele in nearly 33% of all cells whereas no CRISPR/Cas9-induced mutations in predicted off-target loci were identified. Corrected BOECs could be cultured in cell mixtures but demonstrated impaired clonal survival. In contrast, CCM1-deficient BOECs displayed increased resistance to stress-induced apoptotic cell death and could be clonally expanded to high passages. When cultured together, CCM1-deficient BOECs largely replaced corrected as well as heterozygous BOECs.
CONCLUSION: We here demonstrate that a non-viral CRISPR/Cas9 approach can not only be used for gene knockout but also for precise gene correction in hard-to-transfect endothelial cells (ECs). Comparing patient-derived isogenic CCM1+/+ , CCM1+/- , and CCM1-/- ECs, we show that the inactivation of the second allele results in clonal evolution of ECs lacking CCM1 which likely reflects the initiation phase of CCM genesis.

PMID: 31124307 [PubMed – indexed for MEDLINE]