Simultaneous inhibition of DNA-PK and PolÏ´ improves integration efficiency and precision of genome editing.

Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of unintended mutations represent major limitations for genome editing applications caused by the interplay with DNA double-strand break repair pathways. To address this, we conduct a large-scale compound library screen to identify targets for enhancing targeted genome insertions. Our study reveals DNA-dependent protein kinase (DNA-PK) as the most effective target to improve CRISPR/Cas9-mediated insertions, confirming previous findings. We extensively characterize AZD7648, a selective DNA-PK inhibitor, and find it to significantly enhance precise gene editing. We further improve integration efficiency and precision by inhibiting DNA polymerase theta (PolÏ´). The combined treatment, named 2iHDR, boosts templated insertions to 80% efficiency with minimal unintended insertions and deletions. Notably, 2iHDR also reduces off-target effects of Cas9, greatly enhancing the fidelity and performance of CRISPR/Cas9 gene editing.

A high-throughput functional assay for characterization of gamma-aminobutyric acid(A) channel modulators using cryopreserved transiently transfected cells.

The ionotropic -aminobutyric acid (GABA)A receptors are an important family of drug targets for a variety of neurological and psychiatric disorders. Selective modulation of certain subtypes of the receptor could lead to novel or improved therapies. However, the discovery of subtype-selective compounds has been hampered by the lack of a high-throughput functional assay and the difficulty in establishing stable cell lines expressing GABAA receptor in a proper subunit composition. To meet drug discovery need we developed a fluorescent imaging plate reader(FLIPR)-based membrane potential assay with sufficient robustness and reproducibility for use in a high-throughput format. Two major subtypes of GABAA receptor were used: GABAA1 and GABAA2, which are composed of (alpha1)2(beta2)2gama2 and (alpha1)2(beta3)2gama2, respectively. We expressed the receptors by transiently co-transfecting cells with the three subunit DNAs in separate constructs, and by controlling the ratio of the DNA amount for each subunit transfected we forced the cells to express GABAA receptors in a pharmacologically relevant form. A large batch of transfected human embryonic kidney 293 cells were cryopreserved and used to screen and evaluate GABAA modulators.In these cells, agonist activation of GABAA receptor resulted in Cl- efflux and membrane depolarization, which was detected by FLIPR as an increase in fluorescence signal. Based on our characterization of several known GABAA modulators and a test set of compounds known to bind to the GABAA benzodiazepine site, we have demonstrated the validity and utility of this assay for discovery of novel pharmacological agents acting at GABAA receptors.