Genome-scale engineering for systems and synthetic biology

Abstract

Genome-modification technologies enable the rational engineering and perturbation of biological systems. Historically, these methods have been limited to gene insertions or mutations at random or at a few pre-defined locations across the genome. The handful of methods capable of targeted gene editing suffered from low efficiencies, significant labor costs, or both. Recent advances have dramatically expanded our ability to engineer cells in a directed and combinatorial manner. Here, we review current technologies and methodologies for genome-scale engineering, discuss the prospects for extending efficient genome modification to new hosts, and explore the implications of continued advances toward the development of flexibly programmable chasses, novel biochemistries, and safer organismal and ecological engineering.

Figure 1

A historical timeline of selected advances leading to genome-scale engineering.

Figure 2

Foundational genome engineering tools and approaches are needed to extend single site genetic perturbations of a single genome to multiple changes across many genomes.

Figure 3

Mechanisms of various targeted genome-modification tools. Integrases can insert a circular donor construct into a recognition site on the genome. Recombinase-mediated cassette exchange (RMCE) involves the replacement of a target sequence flanked with recognition sites with a donor cassette flanked by compatible sites. Homologous recombination using double-stranded DNA cassettes enables programmable target replacement using RecA or RecET-like machinery, which can be stimulated via site-specific cleavage using zinc-finger, CRISPR, or TAL nucleases. Group II introns and insertional elements can be designed to insert into site-specific genome targets. Oligo-mediated allelic replacement incorporates short oligonucleotides into the lagging strand of replicating DNA, which are then resolved upon subsequent cell divisions to inherit the designed mutation.

Figure 4

Strategies for genome-scale engineering by multiplexed in vivo editing or de novo synthesis. Multiplex genome editing enables construction of new genomes via living intermediates for rapid design and test cycles. De novo genome synthesis can build synthetic designs that are drastically different from natural genomes.

Figure 5

Current approaches to genome-scale engineering for building (editing versus synthesis) and testing (screening versus selection) genomes, when considering number of genetic perturbations against total number of mutants generated.

Figure 6

Toward the construction of a flexibly programmable chassis. Genome minimization reduces biological complexity and redundancy. Whole-genome codon remapping enables orthogonal information encoding and expansion of the genetic code. De novo genome synthesis and reconstitution from natural genomes enables creation of semi-synthetic and chimeric genomes with new and hybrid features. Whole-genome redesign and rewiring of regulatory systems enable new synthetic circuitries that are easier to design and model.