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.

ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts.

Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.