Precise in vivo genome editing via single homology arm donor mediated intron-targeting gene integration for genetic disease correction

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In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of premature-aging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications.

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We are grateful to M. Schwarz and P. Schwarz for administrative help; D. O’Keefe and S. Tsuji for help with manuscript preparation; M. Kay and Z.Y. Chen for sharing experimental materials; J. Naughton and J. Marlett for AAV production; K. Peterson and Y. Gu for helping the measurement of heart rate; U. Manor and K. Diffenderfer for imaging; K. McIntyre for mouse histology processing and J. Li for helping the molecular work; K. Sumiyama for data analysis. M.Y. was partially supported by 2016 Salk Women & Science Special Award. K.S. was supported by JSPS KAKENHI (15K21762 and 18H04036), Takeda Science Foundation, The Uehara Memorial Foundation, National Institutes of Natural Sciences (BS291007), The Sumitomo Foundation (170220), The Naito Foundation, The Kurata Grants (1350), Mochida Memorial Foundation, and The Inamori Foundation. This research was supported by Guangdong Provincial Key Laboratory of Genome Read and Write (No. 2017B030301011), Guangdong Provincial Academician Workstation of BGI Synthetic Genomics (No. 2017B090904014) and Shenzhen Peacock Plan (No. KQTD20150330171505310). J.C.I.B. was supported by The Leona M. and Harry B. Helmsley Charitable Trust (2012-PG-MED002), the G. Harold and Leila Y. Mathers Charitable Foundation, NIH (R01HL123755 and 5 DP1 DK113616), The Progeria Research Foundation, The Glenn Foundation, KAUST, The Moxie Foundation, Fundación Dr. Pedro Guillen, AFE and Universidad Católica San Antonio de Murcia (UCAM).

Author information

K.S., M.Y., R.H.-B., and J.C.I.B. conceived the project and designed experiments. K.S., M.Y., R.H.-B. and E.A. constructed plasmids and minicircles. M.Y. and R.H.-B. performed work on primary neurons. K.S., M.Y., R.H.-B., R.D.S., E.A., M.S., and A.N.N. performed genomic DNA and RNA analyses. M.Y., F.H., M.K., T.H., and C.R.E. performed phenotypic analyses of progeria mice. M.Y. performed AAV IV injection in neonatal mice. Z.L., C.W., J.G., Y.Y., Y.G. and K.Z. performed deep sequencing and analyzed data. K.S., M.Y., P.R. and A.O. performed ECG recordings. K.S., E.N.D., J.M.C., P.M., P.G., C.L.-O., G.-H.L., and J.C.I.B. supervised and contributed to the direction of the project. K.S., M.Y., R.H.-B., P.R., J.W. and J.C.I.B. wrote the manuscript with input from all the authors.

Correspondence to Keiichiro Suzuki or Juan Carlos Izpisua Belmonte.

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