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

Mol Genet Genomic Med. 2019 Jul;7(7):e00755. doi: 10.1002/mgg3.755. Epub 2019 May 23.


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.

Keywords: CCM1; CRISPR/Cas9; blood outgrowth endothelial cells; cerebral cavernous malformation; mutation correction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • CRISPR-Cas Systems
  • Cells, Cultured
  • Endothelial Cells / metabolism*
  • Gene Editing / methods*
  • Genetic Therapy / methods
  • Hemangioma, Cavernous, Central Nervous System / genetics*
  • Hemangioma, Cavernous, Central Nervous System / therapy
  • Humans
  • KRIT1 Protein / genetics*
  • KRIT1 Protein / metabolism
  • Proof of Concept Study


  • KRIT1 Protein
  • KRIT1 protein, human