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.

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Genome‐scale engineering for systems and synthetic biology

Much of what is known about mammalian cell regulation has been achieved with the aid of transiently transfected cells. However, overexpression can violate balanced gene dosage, affecting protein folding, complex assembly and downstream regulation. To avoid these problems, genome engineering technologies now enable the generation of stable cell lines expressing modified proteins at (almost) native levels.

The transience of transient overexpression

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2013.11.035

Recent advances in mammalian protein production

Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges.

CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production.

Recombinase-mediated cassette exchange (RMCE) - a rapidly-expanding toolbox for targeted genomic modifications.

The availability of site-specific recombinases has revolutionized the rational construction of cell lines with predictable properties. Early efforts were directed to providing pre-characterized genomic loci with a single recombinase target site that served as an address for the integration of vectors carrying a compatible tag. Efficient procedures of this type had to await recombinases like ΦC31, which recombine attP and attB target sites in a one-way reaction — at least in the cellular environment of the higher eukaryotic cell. Still these procedures lead to the co-introduction of prokaryotic vector sequences that are known to cause epigenetic silencing. This review illuminates the actual status of the more advanced recombinase-mediated cassette exchange (RMCE) techniques that have been developed for the major members of site-specific recombinases (SR), Flp, Cre and ΦC31. In RMCE the genomic address consists of a set of heterospecific recombinase target (RT-) sites permitting the exchange of the intervening sequence for the gene of interest (GOI), as part of a similar cassette. This process locks the GOI in place and it is ‘clean’ in the sense that it does not co-introduce prokaryotic vector parts nor does it leave behind a selection marker.

Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology.

Novel Tag-and-Exchange (RMCE) Strategies Generate Master Cell Clones with Predictable and Stable Transgene Expression Properties

PARP-1 Expression in the Mouse is Controlled by an Autoregulatory Loop: PARP-1 Binding to an Upstream S/MAR Element and to a Novel Recognition Motif in its Promoter Suppresses Transcription

Junhua Qiao

Papers

Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges.

Recombinase-mediated cassette exchange (RMCE) - a rapidly-expanding toolbox for targeted genomic modifications.

Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology.

Novel Tag-and-Exchange (RMCE) Strategies Generate Master Cell Clones with Predictable and Stable Transgene Expression Properties

PARP-1 Expression in the Mouse is Controlled by an Autoregulatory Loop: PARP-1 Binding to an Upstream S/MAR Element and to a Novel Recognition Motif in its Promoter Suppresses Transcription