Precise CCM1 gene correction and inactivation in patient-derived endothelial cells: Modeling Knudson's two-hit hypothesis in vitro.
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.
['Spiegler|Stefanie|S|0000-0002-7405-2554', 'Rath|Matthias|M|', 'Much|Christiane D|CD|', 'Sendtner|Barbara S|BS|', 'Felbor|Ute|U|']
D000328:Adult D064113:CRISPR-Cas Systems D002478:Cells, Cultured D042783:Endothelial Cells D000072669:Gene Editing D015316:Genetic Therapy D020786:Hemangioma, Cavernous, Central Nervous System D006801:Humans D000075927:KRIT1 Protein D000075082:Proof of Concept Study
CCM1 CRISPR/Cas9 cerebral cavernous malformation blood outgrowth endothelial cells mutation correction