Improved bacterial recombineering by parallelized protein discovery.

Autor:	Wannier TM; Department of Genetics, Harvard Medical School, Boston, MA 02115; timothy_wannier@hms.harvard.edu gchurch@genetics.med.harvard.edu., Nyerges A; Department of Genetics, Harvard Medical School, Boston, MA 02115.; Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged HU-6726, Hungary., Kuchwara HM; Department of Genetics, Harvard Medical School, Boston, MA 02115., Czikkely M; Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged HU-6726, Hungary., Balogh D; Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged HU-6726, Hungary., Filsinger GT; Department of Genetics, Harvard Medical School, Boston, MA 02115., Borders NC; Department of Genetics, Harvard Medical School, Boston, MA 02115., Gregg CJ; Department of Genetics, Harvard Medical School, Boston, MA 02115., Lajoie MJ; Department of Genetics, Harvard Medical School, Boston, MA 02115., Rios X; Department of Genetics, Harvard Medical School, Boston, MA 02115., Pál C; Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged HU-6726, Hungary., Church GM; Department of Genetics, Harvard Medical School, Boston, MA 02115; timothy_wannier@hms.harvard.edu gchurch@genetics.med.harvard.edu.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2020 Jun 16; Vol. 117 (24), pp. 13689-13698. Date of Electronic Publication: 2020 May 28.
DOI:	10.1073/pnas.2001588117
Abstrakt:	Exploiting bacteriophage-derived homologous recombination processes has enabled precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, multiplex automated genome engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however, currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, efficient SSAPs must first be found. Here we introduce a high-throughput method for SSAP discovery that we call "serial enrichment for efficient recombineering" (SEER). By performing SEER in Escherichia coli to screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5- to 10-fold in E. coli , while PapRecT enables efficient recombineering in Pseudomonas aeruginosa , a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria. Competing Interests: Competing interest statement: G.M.C. has related financial interests in EnEvolv, GRO Biosciences, and 64-x. G.M.C., C.J.G., M.J.L., and X.R. have submitted a patent application relating to pieces of this work (WO2017184227A2). T.M.W., G.T.F., and G.M.C. have submitted a patent application related to the improved single-stranded DNA-annealing proteins variants referenced here. A.N. and C.P. have submitted a patent application related to directed evolution with random genomic mutations (DIvERGE) (PCT/EP2017/082574 [WO2018108987] Mutagenizing Intracellular Nucleic Acids).
Databáze:	MEDLINE
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