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- Glasbey, JC, et al.
(author)
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- 2021
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swepub:Mat__t
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| 2. |
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| 3. |
- Wimberger, Sandra, 1987, et al.
(author)
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Simultaneous inhibition of DNA-PK and Pol ϴ improves integration efficiency and precision of genome editing
- 2023
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In: Nature Communications. ; 14:1
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Journal article (peer-reviewed)abstract
- 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 (Pol ϴ). 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. Low efficiency of target DNA integration remains a challenge in genome engineering. Here the authors perform large-scale compound library and genetic screens to identify targets that enhance gene editing: they see that combined DNA-PK and Pol ϴ inhibition with potent compounds increases editing efficiency and precision.
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| 4. |
- Peterka, M., et al.
(author)
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Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing
- 2022
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1
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Journal article (peer-reviewed)abstract
- 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. Prime editing is a next-generation approach for precision genome engineering. Here the authors design a nuclease-based prime editor that leverages DNA repair pathways for targeted genomic insertions.
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