Abstract

The role of genetics, predisposing to lung disease and influencing patient-specific drug responses, is gaining attention. However, further research is hampered by the complexity of generating relevant genetic models.

Patient-derived induced-pluripotent stem cells (iPSCs) are powerful tools for disease modelling and drug testing. Genetic engineering technologies, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, have successfully corrected patient-derived iPSCs, but efficiency remains an important challenge.

Based on our previous work achieving CRISPR-Cas9 mediated gene-edited iPSCs in ~ 3 weeks with 1-10% efficiency, we further optimised transfection, guideRNA/nuclease ratio and screening strategies for CRISPR-Cas9 mediated iPSC gene-editing, to increase this efficiency. We also explored cytidine and adenine base editors that enable the conversion of a single base to another (C:G-to-T:A and T:A-to-C:G) without the need to introduce DNA double-strand breaks. Using idiopathic pulmonary fibrosis (IPF) patient-derived iPSCs carrying a heterozygous mutation, we demonstrated that base editing overperformed our optimised CRISPR-Cas9 mediated gene-editing achieving 80% editing efficiency and requiring fewer clonal isolation events. Human iPSCs modified by base editors maintained a normal karyotype, pluripotency marker expression and generated alveolar type 2 (AT2) cells, highly relevant to study IPF in a patient-specific manner.

Our optimised CRISPR-Cas9 mediated and base editing methods will improve patient-specific drug testing, expand our knowledge on the role of genetics in respiratory diseases and help building the next generation of gene-editing based therapeutics.