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.

Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing.

Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing leads to increased levels of by-products, it can rescue pegRNAs that perform poorly with a nickase-based prime editor. We also present a small molecule approach that yields increased product purity of PEn editing. Next, we develop a homology-independent PEn editing strategy, which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.

Interleukin-19 blockade attenuates collagen-induced arthritis in rats.

OBJECTIVES: RA is the most common form of inflammatory arthritis. IL-19 acts as a pro-inflammatory cytokine involved in the pathogenesis of RA. We investigated whether anti-IL-19 antibody treatment would modulate the severity of the disease in a CIA rat model. METHODS: We generated a CIA model by immunizing rats with bovine type II collagen. CIA rats were s.c. treated with anti-IL-19 antibody 1BB1. The effects of 1BB1 on CIA rats were determined by hind-paw thickness, severity score, bone destruction, BMD and cytokine production, which were evaluated using radiological scans, micro-CT, real-time quantitative PCR and ELISA. To analyse gene regulation by IL-19, rat synovial fibroblasts (SFs) were isolated and analysed for the expression of TNF-α, IL-1ß and RANK ligand (RANKL). RESULTS: In vivo, IL-19 was highly expressed in the synovial tissue and SFs isolated from CIA rats. 1BB1 significantly ameliorated the severity of arthritis by decreasing hind-paw thickness and swelling; prevented bone destruction and bone loss; inhibited the expression of TNF-α, IL-1ß, IL-6 and RANKL in synovial tissue; and decreased the production of IL-6 in serum. In vitro, IL-19-induced TNF-α, IL-1ß, IL-6 and RANKL expression in CIA SFs. CONCLUSIONS: Specifically blocking IL-19 inhibited pro-inflammatory cytokine production and prevented bone destruction in CIA rats. These findings provide evidence that IL-19 is a novel target, and that anti-IL-19 antibody may be a potential target to ameliorate the severity of RA.