Low RT-based Genome Editing Fidelity in Mouse Hepatocytes: Challenges and Solutions

Dunyak, M. T.; Hanna, P.; Nan, A. X.; von Stetina, J.; Pokharel, R.; O'Hara, M.; Estes, B. J.; Zheng, K.; Svenson, S.; Andresen, J.; Li, W.; Agrawal, M.; Hughes, C.; Spicer, M.; Choudhary, V.; Kelley, L.; Wang, J.; Meagher, M.; Xie, J.; Mukherjee, S.; Finn, J. D.

doi:10.1101/2024.10.31.621282

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bioRxiv · 10.1101/2024.10.31.621282

Low RT-based Genome Editing Fidelity in Mouse Hepatocytes: Challenges and Solutions

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Abstract

Abstract/SummaryIntegrase-mediated Programmable Genomic Integration (I-PGI) uses a Cas9 nickase (nCas9) with a reverse transcriptase (RT), to write a large serine integrase (LSI) target site (attB/P, here called "beacon") in a programmed location. Co-delivery of the LSI and a DNA template containing the cognate recognition site results in precise integration of the template in a specific genomic location. While we were able to achieve high-fidelity beacon placement in a range of primate cycling and non-dividing cells, when translating our technology into an in vivo rodent model (liver) we surprisingly observed very low beacon fidelity, with the vast majority of beacons being unsuitable for integration. This phenomenon was independent of mouse strain, but was specific to non-dividing cells, as a cycling mouse hepatocyte cell line (Hepa1-6) demonstrated very high levels of fidelity. To address this issue we utilized neonatal mice, which have a much higher proportion of proliferating hepatocytes than adult mice. This resulted in a significant increase in the placement of high-fidelity beacons, and achieved functional gene expression after I-PGI in a therapeutically relevant target site. In an alternate approach, we engineered transgenic mice with intact beacons placed in specific genomic locations, allowing us to optimize integrase and DNA template dosing and kinetics. In summary, we have identified a previously undescribed challenge when using RT-based editing to write long sequences (~40 bp) in non-dividing rodent hepatocytes. This phenomenon was specific to rodents and was not observed in primate dividing or non-dividing cells. This previously unidentified challenge using RTs will limit the use of I-PGI in mouse models, however here we describe two methods that address this issue.

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Dunyak, M. T., Hanna, P., Nan, A. X., von Stetina, J., Pokharel, R., O'Hara, M., Estes, B. J., Zheng, K., Svenson, S., Andresen, J., Li, W., Agrawal, M., Hughes, C., Spicer, M., Choudhary, V., Kelley, L., Wang, J., Meagher, M., Xie, J., Mukherjee, S., Finn, J. D.. 2024-11-03. Low RT-based Genome Editing Fidelity in Mouse Hepatocytes: Challenges and Solutions. https://doi.org/10.1101/2024.10.31.621282

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