Genomes by design

Abstract

Next-generation DNA sequencing has revealed the complete genome sequences of numerous organisms, establishing a fundamental and growing understanding of genetic variation and phenotypic diversity. Engineering at the gene, network and whole-genome scale aims to introduce targeted genetic changes both to explore emergent phenotypes and to introduce new functionalities. Expansion of these approaches into massively parallel platforms establishes the ability to generate targeted genome modifications, elucidating causal links between genotype and phenotype, as well as the ability to design and reprogramme organisms. In this Review, we explore techniques and applications in genome engineering, outlining key advances and defining challenges.

Figure 1. Genes, networks and genomes by design

a | Both Darwinian evolution and rational genome engineering rely on the introduction of mutations to traverse a genotype space and explore the resultant changes in cellular phenotype. The high-dimensional nature of genotype landscapes presents a key challenge in resolving causal genotype–phenotype relationships and engineering organisms with prescribed behaviours. Genome engineering seeks to leverage existing knowledge, modern DNA manipulation techniques and evolution to search through complex genotype landscapes more efficiently. b | The genome can first be thought of as a physical collection of its constituent DNA elements. These constituent parts can be conceptualized as nodes in a network in which edges represent functional connections (for example, genes involved in the same cellular process). Networks can range in size from small collections of genes in biosynthetic clusters to the genome-scale network of all genes and cellular processes. The space of all possible genotypes can be visualized as a phenotypic landscape where each genotype is associated with a particular level of performance with respect to various phenotypic traits. Manipulation of the genome can alter the connections in cellular networks or add new nodes. These new networks can represent a new location in the phenotype landscape or alter the phenotype landscape itself. Changes at the gene and small-network level enable exploration of broadened phenotype landscapes, whereas large-scale genome modification can yield disruptive and novel fitness landscapes.

Figure 2. Nuclease-mediated genome engineering

a | Nuclease-based gene editing using zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or CRISPR–Cas targeting induces double-stranded breaks that are repaired in a template-dependent (homology-directed repair) or -independent (non-homologous end-joining) manner. b | Modified CRISPR–Cas systems have been used as transcriptional activators by stabilizing RNA polymerase (RNAP) binding at specified sites. c | Transcriptional repressors that use modified CRISPR–Cas to block transcriptional initiation or elongation have also been developed. d | Targeting CRISPR–Cas cleavage to sequences that are unique to a specific genome results in the selective removal of that genome from a bacterial population. e | In the context of recombination-mediated genome engineering, targeting CRISPR–Cas cleavage to the wild-type sequence has been used to remove unaltered cells and enrich for mutations. f | Gene drives using CRISPR–Cas systems cut homologous chromosomes and promote their own incorporation via homology-directed repair. Placement of constructs near genes of interest can facilitate their spread throughout a population. PAM, protospacer adjacent motif.

Figure 3. Gene and network-scale recombineering

a|Recombineering (recombination-mediated genetic engineering) uses cellular homologous recombination enhanced by phage proteins to recombine single-stranded DNA (ssDNA) or double-stranded DNA into the genome. This enables site-specific introduction of mutations ranging in resolution from single base pair changes to the introduction of entire genes. b | The SCRIBE (Synthetic Cellular Recorders Integrating Biological Events) analogue memory system uses reverse transcriptase (purple) to produce ssDNA in vivo, which is then used in recombineering. SCRIBE is able to introduce heritable genetic changes (*) in response to a predetermined stimulus. These heritable changes act as a cellular memory of exposure to the stimulus. The fraction of cells containing this mutation serves as an approximation of the cumulative stimulus input function. c | Multiplex automated genome engineering (MAGE) uses the introduction and incorporation of synthetic ssDNA at the replication fork to introduce desired mutations. Mutations can be targeted to multiple residues in a single gene, multiple genes in a pathway, or across the genome. d | Trackable multiplex recombineering (TRMR) utilizes recombineering and high-throughput profiling to introduce mutations and to identify variants that provide a fitness benefit under different conditions. Each recombineering cassette contains a selectable marker, targeting homology domain, and a sequence to increase or decrease gene expression. Sequencing of genetic barcodes associated with each mutation reveals the resultant enrichment of mutations that confer a fitness benefit.

Figure 4. Refactored pathways and minimal genomes

a | There are numerous approaches to the transfer of gene clusters. Biosynthetic clusters can be cloned from their native organism and transferred to another organism for heterologous expression. For organisms that are recalcitrant to laboratory culture techniques, gene sequences of interest can be identified from genomic or metagenomic databases and subsequently synthesized and introduced into a production organism. The native regulatory elements of a heterologous pathway may not be optimal for expression in the host organism. Refactoring approaches seek to rebuild the pathways using well-characterized modular regulatory elements and removing all native regulation. b | Recombineering (recombination-mediated genetic engineering) was used to delete up to 15% of the wild-type Escherichia coli K12 genome, including insertion sequence (IS) elements. This led to a more stable and streamlined genetic architecture that manifested itself in altered cellular phenotypes. c | The de novo synthesis of the Mycoplasma mycoides genome utilized synthetic DNA oligonucleotides assembled first in vitro and then in Saccharomyces cerevisiae. Following completion of the ~1 Mb genome, the whole construct was transplanted to Mycoplasma capricolum, yielding a viable organism. BAC, bacterial artificial chromosome; RBS, ribosome-binding site.

Figure 5. Genome recoding and new biological functions

a | The genomically recoded organism was constructed by first using multiplex automated genome engineering (MAGE) to convert TAG stop codons to TAA in a panel of strains. Conjugative assembly genome engineering (CAGE) was then used for the hierarchical assembly of these partially recoded genomes into a fully recoded genome in which all 321 TAG stop codons are reassigned. Release factor 1 (RF1) was then deleted in order to establish TAG as an open codon for the incorporation of non-standard amino acids (nsAAs). b | The addition of an orthogonal translation system capable of charging a nsAA to a TAG-recognizing tRNA enables the genetic encoding of nsAA-incorporating proteins. c | The reassignment of TAG from a stop codon can render TAG-containing or -terminating genes non-functional in a different organism. This reduces the probability of horizontal gene transfer (HGT) between recoded and natural organisms. d | The incorporation of TAG codons in essential genes provides a method for the biocontainment of recoded organisms by linking its viability to the presence of an exogenous nsAA. aaRS, aminoacyl-tRNA synthetase.

Figure 6. Design principles and assembly: synthetic yeast genome project

a | Design principles in the synthetic yeast genome project include: wild-type phenotypes, unburdened and stabilized chromosomes via the removal of numerous types of genetic elements, and increased genetic flexibility through recoding of all TAG codons to TAA and introduction of symmetrical loxP (loxPsym) sites after non-essential genes. b | Synthetic DNA assembled in vitro is incorporated into the genome in a piece-wise manner, permitting phenotype characterization of each hierarchical segment. Induction of Cre recombinase drives activity of loxPsym, leading to chromosomal rearrangements that eliminate and translocate segments, enabling rapid exploration of dramatically altered genotypes. ORF, open reading frame; UTR, untranslated region.