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

Autor: Durrant MG; Arc Institute, Palo Alto, CA, USA.; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.; Department of Genetics, Stanford University, Stanford, CA, USA., Fanton A; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.; University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, Berkeley, CA, USA., Tycko J; Department of Genetics, Stanford University, Stanford, CA, USA., Hinks M; Department of Bioengineering, Stanford University, Stanford, CA, USA., Chandrasekaran SS; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.; University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, Berkeley, CA, USA., Perry NT; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.; University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, Berkeley, CA, USA., Schaepe J; Department of Bioengineering, Stanford University, Stanford, CA, USA., Du PP; Department of Genetics, Stanford University, Stanford, CA, USA.; Cancer Biology Program, Stanford University, Stanford, CA, USA., Lotfy P; Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies, La Jolla, CA, USA., Bassik MC; Department of Genetics, Stanford University, Stanford, CA, USA. bassik@stanford.edu., Bintu L; Department of Bioengineering, Stanford University, Stanford, CA, USA. lbintu@stanford.edu., Bhatt AS; Department of Genetics, Stanford University, Stanford, CA, USA. asbhatt@stanford.edu.; Department of Medicine (Hematology), Stanford University, Stanford, CA, USA. asbhatt@stanford.edu., Hsu PD; Arc Institute, Palo Alto, CA, USA. pdhsu@berkeley.edu.; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.; Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies, La Jolla, CA, USA. pdhsu@berkeley.edu.; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.; Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.
Jazyk: angličtina
Zdroj: Nature biotechnology [Nat Biotechnol] 2023 Apr; Vol. 41 (4), pp. 488-499. Date of Electronic Publication: 2022 Oct 10.
DOI: 10.1038/s41587-022-01494-w
Abstrakt: 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.
(© 2022. The Author(s).)
Databáze: MEDLINE
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