A versatile platform strain for high-fidelity multiplex genome editing

Abstract

21Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the 22 engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have 23 enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed 24 mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date 25 been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of 26 inducible transcriptional regulators in these strains also prevents concurrent control of genome 27 engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, 28BioDesignER, which incorporates (1) a refactored λ-Red recombination system that reduces toxicity 29 and accelerates multi-cycle recombination, (2) genetic modifications that boost recombination 30 efficiency, and (3) four independent inducible regulators to control engineered functions. These 31 modifications resulted in single-cycle recombineering efficiencies of up to 25% with a seven-fold 32 increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To 33 facilitate genome engineering in BioDesignER, we have curated eight context-neutral genomic loci,34 termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is 35 a platform to develop and optimize engineered cellular functions and can serve as a model to implement 36 comparable recombination and regulatory systems in other bacteria. 37 INTRODUCTION 38The design-build-test (DBT) cycle is a common paradigm used in engineering disciplines. Within the 39 context of synthetic biology it is employed to engineer user-defined cellular functions for applications 40 such as metabolic engineering, biosensing, and therapeutics (1, 2). The rapid prototyping of engineered 41 functions has been facilitated by advances in in vitro DNA assembly, and plasmids have traditionally 42 been used to implement designs in vivo given their ease-of-assembly and portability. However, for 43 deployment in contexts beyond the laboratory such as large-scale industrial bioprocesses or among 44 complex microbial communities, plasmid-based circuits suffer from multiple limitations: high intercellular 45 variation in gene expression, genetic instability from random partitioning of plasmids during cell division, 46 and plasmid loss in environments for which antibiotic use could disrupt native microbial communities or 47 is economically infeasible (3, 4). These shortcomings can be ameliorated once a design is transferred 48 from a plasmid to the host genome, which offers improved genetic stability and lower expression 49 variation (5) along with reduced metabolic load (6). However, behaviors optimized for plasmid contexts 50 often do not map predictably to the genome. As such, building and testing designs directly on the 51 genome can reduce the DBT cycle time and facilitate engineering cellular programs for complex 52 e...

Species- and site-specific genome editing in complex bacterial communities

“…Putative safe genome integration loci, referred to as Safe Sites, were identified in K. michiganensis (GCF_002090205.1) and P. simiae (GCF_000698275.1) following previously reported methods 31 . Specifically, all intergenic regions between two convergently transcribed genes were rank ordered by size and filtered to remove those containing predicted RNA features 32,33 , those inside or adjacent to a putative mobile genetic element, and those flanked by at least one likely essential gene (in which no insertions were obtained in a high coverage genome-wide transposon mutant library) or genes exhibiting fitness defects in any previously screened conditions 34 .…”

A standardized genome architecture for bacterial synthetic biology (SEGA)

“…This difference may be a result of transcriptional silencing of the ykgH locus through protein occupancy 57,61 . The 11 integration sites explored here do not necessarily represent optimized expression loci: recent studies have attempted to identify and characterize genomic integration sites with minimal disruption of the host expression that might be better suited for further optimization 32,60,62 and the relative expression strength of a genome location can depend on the growth media and carbon source 60 . Another recent study comprehensively characterizes multiple areas of transcriptional silencing and enhancement 57 .…”

Tunable dynamics in a multi-strain transcriptional pulse generator

“…Strains DZ06, DZ07 and DZ08 were modified with a unique 25bp barcode for identification using amplicon sequencing. This 25bp barcode, along with surrounding regions from the knock in cassette are located on the genome at the SS3 locus ( 35 ). Primers were designed to bind to regions flanking the unique barcodes such all three barcodes could be amplified using one primer pair in a single PCR reaction.…”