Improvements in DNA-editing technologies have enabled engineering of microorganisms at the gene, network and genome level, helping to elucidate causal links between genotypes and phenotypes, and facilitating the targeted exploration of complex phenotype landscapes.
DNA sequencing and genome engineering are synergistic technologies: understanding genetic and phenotypic diversity establishes a platform for pursuing many goals in biological design.
Multiplex genome modification techniques have enabled combinatoric genomic variations across populations, exploring large numbers of genotype–phenotype landscapes in living cells.
The combination of DNA synthesis and in vivo genome editing establishes the ability to fundamentally redesign systems from refactored pathways to whole-genome recoding.
The future of genome editing will require the combination of de novo synthesis and in vivo genome evolution integrated with functional screens and selections to isolate variants exhibiting a desired phenotype.
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.
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The authors thank M. Lajoie, the Isaacs laboratory and the anonymous reviewers for helpful comments on this paper. A.D.H. acknowledges support from US National Cancer Institute (NCI) award 1F30CA196191-01. P.M. is supported by the Raymond and Beverly Sackler Institute for Biological, Physical and Engineering Sciences. F.J.I. gratefully acknowledges support from the US Defense Advanced Research Projects Agency (N66001-12-C-4020, N66001-12-C-4211); US Department of Energy (152339.5055249.100); Gen9 Inc., DuPont Inc., and the Arnold and Mabel Beckman Foundation.
The authors declare no competing financial interests.
- Directed evolution
A process that utilizes mutagenesis and a user-defined selective pressure to develop populations that are enriched for mutants exhibiting a desired function or behaviour.
- Nuclease-based genome editing
The use of engineered nucleases with a DNA-targeting mechanism (for example, a zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) or a clustered regularly interspaced short palindromic repeat (CRISPR)) for targeted mutagenesis in vivo.
A bacterial adaptive immune system comprising clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) endonucleases that has been repurposed for genome editing.
(Recombination-mediated genetic engineering). A technique that utilizes homologous recombination to alter genetic loci using synthetic single-stranded DNA and double-stranded DNA.
Searching and sourcing useful genes from other organisms to specifically confer a desired phenotype (for example, medically or industrially relevant pathways and small molecules).
Reorganization of biological systems or pathways with the goal of improving the ease and predictability of future engineering efforts. This often entails the removal of native regulation, separation of overlapping genetic elements and the use of well-characterized regulatory elements.
- Codon optimization
A multifactorial process that utilizes synonymous codons to reflect the preferred codon frequency in order to optimize protein expression in a target species.
- Gene synthesis
De novo construction of user-defined double-stranded DNA.
- Genetic code expansion
Modification of the genetic code to enable site-directed incorporation of amino acids beyond the canonical 20 into proteins.
- Non-standard amino acid
(nsAA). An amino acid beyond the canonical 20 present in the genetic code. nsAAs generally fall into two classes: post-translational modifications (for example, phosphoserine); and synthetic amino acids that do not exist in nature.
- Orthogonal translation systems
(OTSs). Used in genetic code expansion to link incorporation of a non-standard amino acid (nsAA) to a target codon. These systems are composed of a tRNA that is selectively charged with a given nsAA by a cognate aminoacyl-tRNA synthetase (aaRS). Other possible components of OTSs include mutant elongation factor Tu and ribosomes designed to accommodate the given nsAA.
- Genomically recoded organism
(GRO). An organism in which codons have been reassigned to create an alternate genetic code. For example, an Escherichia coli strain in which all instances of the TAG stop codon have been mutated to TAA and release factor 1 (RF1) has been deleted, enabling the use of TAG as an open coding channel.
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Haimovich, A., Muir, P. & Isaacs, F. Genomes by design. Nat Rev Genet 16, 501–516 (2015). https://doi.org/10.1038/nrg3956
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