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Large-scale discovery of recombinases for integrating DNA into the human genome

Matthew G. Durrant, Alison Fanton, View ORCID ProfileJosh Tycko, Michaela Hinks, View ORCID ProfileSita S. Chandrasekaran, Nicholas T. Perry, Julia Schaepe, Peter P. Du, View ORCID ProfilePeter Lotfy, Michael C. Bassik, View ORCID ProfileLacramioara Bintu, View ORCID ProfileAmi S. Bhatt, Patrick D. Hsu
doi: https://doi.org/10.1101/2021.11.05.467528
Matthew G. Durrant
1Department of Bioengineering, University of California, Berkeley
2Department of Genetics, Stanford University
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Alison Fanton
1Department of Bioengineering, University of California, Berkeley
3University of California, Berkeley—University of California, San Francisco Graduate Program in Bioengineering
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Josh Tycko
2Department of Genetics, Stanford University
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Michaela Hinks
4Department of Bioengineering, Stanford University
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Sita S. Chandrasekaran
1Department of Bioengineering, University of California, Berkeley
3University of California, Berkeley—University of California, San Francisco Graduate Program in Bioengineering
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  • ORCID record for Sita S. Chandrasekaran
Nicholas T. Perry
1Department of Bioengineering, University of California, Berkeley
3University of California, Berkeley—University of California, San Francisco Graduate Program in Bioengineering
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Julia Schaepe
4Department of Bioengineering, Stanford University
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Peter P. Du
5Department of Cancer Biology, Stanford University
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Peter Lotfy
6Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies
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Michael C. Bassik
2Department of Genetics, Stanford University
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  • For correspondence: lbintu@stanford.edu bassik@stanford.edu asbhatt@stanford.edu pdhsu@berkeley.edu
Lacramioara Bintu
4Department of Bioengineering, Stanford University
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  • For correspondence: lbintu@stanford.edu bassik@stanford.edu asbhatt@stanford.edu pdhsu@berkeley.edu
Ami S. Bhatt
2Department of Genetics, Stanford University
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  • For correspondence: lbintu@stanford.edu bassik@stanford.edu asbhatt@stanford.edu pdhsu@berkeley.edu
Patrick D. Hsu
1Department of Bioengineering, University of California, Berkeley
6Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies
7Innovative Genomics Institute, University of California, Berkeley
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  • For correspondence: lbintu@stanford.edu bassik@stanford.edu asbhatt@stanford.edu pdhsu@berkeley.edu
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SUMMARY

Recent microbial genome sequencing efforts have revealed a vast reservoir of mobile genetic elements containing integrases that could be useful genome engineering tools. Large serine recombinases (LSRs), such as Bxb1 and PhiC31, are bacteriophage-encoded integrases that can facilitate the insertion of phage DNA into bacterial genomes. However, only a few LSRs have been previously characterized and they have limited efficiency in human cells. Here, we developed a systematic computational discovery workflow that identifies thousands of new LSRs and their cognate DNA attachment sites by. We validate this approach via experimental characterization of LSRs in human cells, leading to three classes of LSRs distinguished from one another by their efficiency and specificity. We identify landing pad LSRs that efficiently integrate into synthetically installed attachment sites orthogonal to the human genome, human genome-targeting LSRs with computationally predictable pseudosites, and multi-targeting LSRs that can unidirectionally integrate cargos at with similar efficiency and superior specificity to commonly used transposases. LSRs from each category were functionally characterized in human cells, overall achieving up to 7-fold higher plasmid recombination than Bxb1 and genome insertion efficiencies of 40-70% with cargo sizes over 7 kb. Overall, we establish a paradigm for large-scale discovery of microbial recombinases and reconstruction of their target sites directly from microbial sequencing data. This strategy provides a rich resource of over 60 experimentally characterized LSRs that can function in human cells and thousands of additional candidates for large-payload genome editing without exposed DNA double-stranded breaks.

Competing Interest Statement

M.G.D., J.T., A.F., M.C.B., L.B., A.S.B., and P.D.H. are inventors on intellectual property related to this work. P.D.H. is a cofounder of Spotlight Therapeutics and Moment Biosciences and serves on the board of directors and scientific advisory boards, and is a scientific advisory board member to Vial Health and Serotiny.

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  • Author list and references were updated

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-ND 4.0 International license.
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Posted November 09, 2021.
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Large-scale discovery of recombinases for integrating DNA into the human genome
Matthew G. Durrant, Alison Fanton, Josh Tycko, Michaela Hinks, Sita S. Chandrasekaran, Nicholas T. Perry, Julia Schaepe, Peter P. Du, Peter Lotfy, Michael C. Bassik, Lacramioara Bintu, Ami S. Bhatt, Patrick D. Hsu
bioRxiv 2021.11.05.467528; doi: https://doi.org/10.1101/2021.11.05.467528
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Large-scale discovery of recombinases for integrating DNA into the human genome
Matthew G. Durrant, Alison Fanton, Josh Tycko, Michaela Hinks, Sita S. Chandrasekaran, Nicholas T. Perry, Julia Schaepe, Peter P. Du, Peter Lotfy, Michael C. Bassik, Lacramioara Bintu, Ami S. Bhatt, Patrick D. Hsu
bioRxiv 2021.11.05.467528; doi: https://doi.org/10.1101/2021.11.05.467528

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