title: Simultaneous non-contiguous deletions using large synthetic DNA and site-specific recombinases.
abstract: Toward achieving rapid and large scale genome modification directly in a target organism, we have developed a new genome engineering strategy that uses a combination of bioinformatics aided design, large synthetic DNA and site-specific recombinases. Using Cre recombinase we swapped a target 126-kb segment of the Escherichia coli genome with a 72-kb synthetic DNA cassette, thereby effectively eliminating over 54 kb of genomic DNA from three non-contiguous regions in a single recombination event. We observed complete replacement of the native sequence with the modified synthetic sequence through the action of the Cre recombinase and no competition from homologous recombination. Because of the versatility and high-efficiency of the Cre-lox system, this method can be used in any organism where this system is functional as well as adapted to use with other highly precise genome engineering systems. Compared to present-day iterative approaches in genome engineering, we anticipate this method will greatly speed up the creation of reduced, modularized and optimized genomes through the integration of deletion analyses data, transcriptomics, synthetic biology and site-specific recombination.
authors.affiliation: Synthetic Biology and Bioenergy, J. Craig Venter Institute, 9704 Medical Center Dr., Rockville, MD 20850, USA rkrishna@jcvi.org.
authors.forename: Radha
authors.initials: R
authors.lastname: Krishnakumar
authors.affiliation: Synthetic Biology and Bioenergy, J. Craig Venter Institute, 9704 Medical Center Dr., Rockville, MD 20850, USA.
authors.forename: Carissa
authors.initials: C
authors.lastname: Grose
authors.affiliation: Bioinformatics, J. Craig Venter Institute, 9704 Medical Center Dr., Rockville, MD 20850, USA.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Haft
authors.affiliation: Synthetic Biology and Bioenergy, J. Craig Venter Institute, 9704 Medical Center Dr., Rockville, MD 20850, USA.
authors.forename: Jayshree
authors.initials: J
authors.lastname: Zaveri
authors.affiliation: Synthetic Biology and Bioenergy, J. Craig Venter Institute, 9704 Medical Center Dr., Rockville, MD 20850, USA.
authors.forename: Nina
authors.initials: N
authors.lastname: Alperovich
authors.affiliation: Synthetic Biology and Bioenergy, J. Craig Venter Institute, 4120 Torrey Pines Road, La Jolla, CA 92037, USA Synthetic Genomics Inc., 11149 N. Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Gibson
authors.affiliation: Synthetic Biology and Bioenergy, J. Craig Venter Institute, 4120 Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Chuck
authors.initials: C
authors.lastname: Merryman
authors.affiliation: Synthetic Biology and Bioenergy, J. Craig Venter Institute, 9704 Medical Center Dr., Rockville, MD 20850, USA.
authors.forename: John I
authors.initials: JI
authors.lastname: Glass
mesh_terms: D002872:Chromosome Deletion; D004247:DNA / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods; D019426:Integrases / Q000378:metabolism; D011995:Recombination, Genetic; D058615:Synthetic Biology / Q000379:methods
chemical_list: D004247:DNA; C045073:Cre recombinase; D019426:Integrases