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Directed evolution of hyperactive integrases for site specific insertion of transgenes.
Nucleic Acids Res
August 2024
Department of Cell and Molecular Biology, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96814, USA.
The ability to deliver large transgenes to a single genomic sequence with high efficiency would accelerate biomedical interventions. Current methods suffer from low insertion efficiency and most rely on undesired double-strand DNA breaks. Serine integrases catalyze the insertion of large DNA cargos at attachment (att) sites.
View Article and Find Full Text PDFDirected evolution of hyperactive integrases for site specific insertion of transgenes.
bioRxiv
June 2024
Department of Cell and Molecular Biology, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, 96814 USA.
The ability to deliver large transgenes to a single genomic sequence with high efficiency would accelerate biomedical interventions. Current methods suffer from low insertion efficiency and most rely on undesired double-strand DNA breaks. Serine integrases catalyze the insertion of large DNA cargos at attachment (att) sites.
View Article and Find Full Text PDFEfficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing.
Nat Biomed Eng
June 2024
Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
Methods for the targeted integration of genes in mammalian genomes suffer from low programmability, low efficiencies or low specificities. Here we show that phage-assisted continuous evolution enhances prime-editing-assisted site-specific integrase gene editing (PASSIGE), which couples the programmability of prime editing with the ability of recombinases to precisely integrate large DNA cargoes exceeding 10 kilobases. Evolved and engineered Bxb1 recombinase variants (evoBxb1 and eeBxb1) mediated up to 60% donor integration (3.
View Article and Find Full Text PDFMultiplex Functional Characterization of Protein Variant Libraries in Mammalian Cells with Single-Copy Genomic Integration and High-Throughput DNA Sequencing.
Methods Mol Biol
March 2024
Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA.
Sequencing-based, massively parallel genetic assays have enabled simultaneous characterization of the genotype-phenotype relationships for libraries encoding thousands of unique protein variants. Since plasmid transfection and lentiviral transduction have characteristics that limit multiplexing with pooled libraries, we developed a mammalian synthetic biology platform that harnesses the Bxb1 bacteriophage DNA recombinase to insert single promoterless plasmids encoding a transgene of interest into a pre-engineered "landing pad" site within the cell genome. The transgene is expressed behind a genomically integrated promoter, ensuring only one transgene is expressed per cell, preserving a strict genotype-phenotype link.
View Article and Find Full Text PDFHigh fidelity one-pot DNA assembly using orthogonal serine integrases.
Biotechnol J
March 2023
School of Molecular Biosciences, University of Glasgow, Glasgow, UK.
Background: Large serine integrases (LSIs, derived from temperate phages) have been adapted for use in a multipart DNA assembly process in vitro, called serine integrase recombinational assembly (SIRA). The versatility, efficiency, and fidelity of SIRA is limited by lack of a sufficient number of LSIs whose activities have been characterized in vitro.
Methods And Major Results: In this report, we compared the activities in vitro of 10 orthogonal LSIs to explore their suitability for multiplex SIRA reactions.
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