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Programmable large DNA deletion, replacement, integration, and inversion with twin prime editing and site-specific recombinases

Andrew V. Anzalone, Xin D. Gao, Christopher J. Podracky, Andrew T. Nelson, Luke W. Koblan, Aditya Raguram, Jonathan M. Levy, Jaron A. M. Mercer, David R. Liu
doi: https://doi.org/10.1101/2021.11.01.466790
Andrew V. Anzalone
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA
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Xin D. Gao
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA
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Christopher J. Podracky
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA
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Andrew T. Nelson
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA
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Luke W. Koblan
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA
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Aditya Raguram
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA
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Jonathan M. Levy
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA
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Jaron A. M. Mercer
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA
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David R. Liu
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA
2Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA
3Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA
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  • For correspondence: drliu@fas.harvard.edu
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Summary

The targeted deletion, replacement, integration, or inversion of DNA sequences at specified locations in the genome could be used to study or treat many human genetic diseases. Here, we describe twin prime editing (twinPE), a method for the programmable replacement or excision of DNA sequence at endogenous human genomic sites without requiring double-strand DNA breaks. TwinPE uses a prime editor (PE) protein and two prime editing guide RNAs (pegRNAs) that template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, resulting in the replacement of endogenous DNA sequence between the PE-induced nick sites with pegRNA-encoded sequences. We show that twinPE in human cells can perform precise deletions of at least 780 bp and precise replacements of genomic DNA sequence with new sequences of at least 108 bp. By combining single or multiplexed twinPE with site-specific serine recombinases either in separate steps or in a single step, we demonstrate targeted integration of gene-sized DNA plasmids (>5,000 bp) into safe-harbor loci including AAVS1, CCR5, and ALB in human cells. To our knowledge, these results represent the first RNA-programmable insertion of gene-sized DNA sequences into targeted genomic sites of unmodified human cells without requiring double-strand breaks or homology-directed repair. Twin PE combined with recombinases also mediated a 40,167-bp inversion at IDS that corrects a common Hunter syndrome allele. TwinPE expands the capabilities of precision gene editing without requiring double-strand DNA breaks and synergizes with other tools to enable the correction or complementation of large or complex pathogenic alleles in human cells.

Competing Interest Statement

D.R.L. is a consultant and equity holder of Beam Therapeutics, Prime Medicine, Pairwise Plants, and Chroma Medicine, companies that use genome editing or genome engineering technologies. A.V.A., C.J.P., and J.M.L. are currently employees at Prime Medicine. The authors have filed patent applications on twinPE and prime editing through the Broad Institute.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted November 02, 2021.
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Programmable large DNA deletion, replacement, integration, and inversion with twin prime editing and site-specific recombinases
Andrew V. Anzalone, Xin D. Gao, Christopher J. Podracky, Andrew T. Nelson, Luke W. Koblan, Aditya Raguram, Jonathan M. Levy, Jaron A. M. Mercer, David R. Liu
bioRxiv 2021.11.01.466790; doi: https://doi.org/10.1101/2021.11.01.466790
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Programmable large DNA deletion, replacement, integration, and inversion with twin prime editing and site-specific recombinases
Andrew V. Anzalone, Xin D. Gao, Christopher J. Podracky, Andrew T. Nelson, Luke W. Koblan, Aditya Raguram, Jonathan M. Levy, Jaron A. M. Mercer, David R. Liu
bioRxiv 2021.11.01.466790; doi: https://doi.org/10.1101/2021.11.01.466790

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