Systematic discovery of recombinases for efficient integration of large DNA sequences 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

doi:10.1038/s41587-022-01494-w

Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome

Nat Biotechnol. 2023 Apr;41(4):488-499. doi: 10.1038/s41587-022-01494-w. Epub 2022 Oct 10.

Authors

Matthew G Durrant^#^{1

2

3}, Alison Fanton^#^{2

4}, Josh Tycko^#³, Michaela Hinks⁵, Sita S Chandrasekaran^{2

4}, Nicholas T Perry^{2

4}, Julia Schaepe⁵, Peter P Du^{3

6}, Peter Lotfy⁷, Michael C Bassik⁸, Lacramioara Bintu⁹, Ami S Bhatt^{10

11}, Patrick D Hsu^{12

13

14

15

16}

Affiliations

¹ Arc Institute, Palo Alto, CA, USA.
² Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
³ Department of Genetics, Stanford University, Stanford, CA, USA.
⁴ University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, Berkeley, CA, USA.
⁵ Department of Bioengineering, Stanford University, Stanford, CA, USA.
⁶ Cancer Biology Program, Stanford University, Stanford, CA, USA.
⁷ Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies, La Jolla, CA, USA.
⁸ Department of Genetics, Stanford University, Stanford, CA, USA. bassik@stanford.edu.
⁹ Department of Bioengineering, Stanford University, Stanford, CA, USA. lbintu@stanford.edu.
¹⁰ Department of Genetics, Stanford University, Stanford, CA, USA. asbhatt@stanford.edu.
¹¹ Department of Medicine (Hematology), Stanford University, Stanford, CA, USA. asbhatt@stanford.edu.
¹² Arc Institute, Palo Alto, CA, USA. pdhsu@berkeley.edu.
¹³ Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.
¹⁴ Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies, La Jolla, CA, USA. pdhsu@berkeley.edu.
¹⁵ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.
¹⁶ Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.

^# Contributed equally.

Abstract

Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40-75% with cargo sizes over 7 kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Genetic Engineering*
Genome, Human
Genomic Library
Humans
Integrases*
Transfection

Substances

Integrases

Abstract

Publication types

MeSH terms

Substances

Grants and funding