Genome Engineering Strategies Research Articles

High efficiency recombineering in lactic acid bacteria

The ability to efficiently generate targeted point mutations in the chromosome without the need for antibiotics, or other means of selection, is a powerful strategy for genome engineering. Although oligonucleotide-mediated recombineering (ssDNA recombineering) has been utilized in Escherichia coli for over a decade, the successful adaptation of ssDNA recombineering to Gram-positive bacteria has not been reported. Here we describe the development and application of ssDNA recombineering in lactic acid bacteria. Mutations were incorporated in the chromosome of Lactobacillus reuteri and Lactococcus lactis without selection at frequencies ranging between 0.4% and 19%. Whole genome sequence analysis showed that ssDNA recombineering is specific and not hypermutagenic. To highlight the utility of ssDNA recombineering we reduced the intrinsic vancomymycin resistance of L. reuteri >100-fold. By creating a single amino acid change in the d-Ala-d-Ala ligase enzyme we reduced the minimum inhibitory concentration for vancomycin from >256 to 1.5 µg/ml, well below the clinically relevant minimum inhibitory concentration. Recombineering thus allows high efficiency mutagenesis in lactobacilli and lactococci, and may be used to further enhance beneficial properties and safety of strains used in medicine and industry. We expect that this work will serve as a blueprint for the adaptation of ssDNA recombineering to other Gram-positive bacteria.

Multiplexed in Vivo His-Tagging of Enzyme Pathways for in Vitro Single-Pot Multienzyme Catalysis

Protein pathways are dynamic and highly coordinated spatially and temporally, capable of performing a diverse range of complex chemistries and enzymatic reactions with precision and at high efficiency. Biotechnology aims to harvest these natural systems to construct more advanced in vitro reactions, capable of new chemistries and operating at high yield. Here, we present an efficient Multiplex Automated Genome Engineering (MAGE) strategy to simultaneously modify and co-purify large protein complexes and pathways from the model organism Escherichia coli to reconstitute functional synthetic proteomes in vitro. By application of over 110 MAGE cycles, we successfully inserted hexa-histidine sequences into 38 essential genes in vivo that encode for the entire translation machinery. Streamlined co-purification and reconstitution of the translation protein complex enabled protein synthesis in vitro. Our approach can be applied to a growing area of applications in in vitro one-pot multienzyme catalysis (MEC) to manipulate or enhance in vitro pathways such as natural product or carbohydrate biosynthesis.

From genes to function: theC.elegansgenetic toolbox

This review aims to provide an overview of the technologies which make the nematode Caenorhabditis elegans an attractive genetic model system. We describe transgenesis techniques and forward and reverse genetic approaches to isolate mutants and clone genes. In addition, we discuss the new possibilities offered by genome engineering strategies and next-generation genome analysis tools.

Transgene excision in zebrafish using the phiC31 integrase

Genome engineering strategies employing site-specific recombinases (SSRs) have become invaluable to the study of gene function in model organisms. One such SSR, the integrase encoded by the Streptomyces bacteriophage phiC31, promotes recombination between heterotypic attP and attB sites. In the present study I have examined the feasibility of the use of phiC31 integrase for intramolecular recombination strategies in zebrafish embryos. I report here that (1) phiC31 integrase is functional in zebrafish cells, (2) phiC31 integrase can excise a transgene cassette flanked by an attB and an attP site, analogous to a common use of the Cre/lox SSR system, (3) phiC31 integrase functions in the zebrafish germline, and (4) a phiC31 integrase-estrogen receptor hormone-binding domain variant fusion protein catalyzes attB-attP recombination in zebrafish embryos in a 4-hydroxytamoxifen-dependent manner, albeit less efficiently than phiC31 alone. These features should make this a useful approach for genome manipulations in the zebrafish.

"Cre"-ating mouse mutants-a meeting review on conditional mouse genetics.

So you want to make a mouse mutation to study the function of your favorite gene? Making a simple knockout is just the first step. The future is in complex genome engineering strategies that will allow you to knockout or misexpress your gene when and where you want, make a series of allelic alterations, and rearrange the chromosomal context in which the gene resides. However, undertaking this kind of analysis in mice is time-consuming and expensive. Many such strategies involve multiple components, each of which has to be tried and tested in a living animal. Everyone wants to know which systems work, but everyone hopes someone else will do the necessary groundwork to test them out. No wonder, then, that there was a large and attentive audience at a recent National Cancer Institute-sponsored workshop at Cold Spring Harbor Laboratory on Conditional Genetic Technologies in the Mouse, (August 31–September 2, 1998), all hoping to learn the latest successes in this area. They were treated to a series of talks by expert practioners in the field, who not only presented their success stories but also some of the problems and failures that do not make it into the published literature. Most of the talks and their accompanying slides can be accessed online at http://www.leadingstrand.org/. Although there is enormous potential in this area, the workshop clearly revealed that there is no fully developed, guaranteed successful kit of genetic reagents for creating conditional alterations. One reagent, however, can be considered out of the development phase and into the catalog of standard genetic tools. That reagent is, of course, the site-specific recombinase Cre. The Cre recombinase can excise DNA sequences between two loxP recognition sequences at a very high efficiency either in mammalian cells in culture or in mice. Tissue-specific gene knockout can be achieved by excision of a loxPflanked (‘floxed’) critical region of the gene after expression of Cre in the tissue of interest. Several successful examples of this approach were presented at this meeting and more at the succeeding Mouse Molecular Genetics Meeting. The key is still the development of suitable tissue-specific Cre-expressing lines that combine exquisite specificity with high-level expression. Nonspecific expression can confuse interpretation of the phenotype, especially if expression occurs early in the embryo, leading to excision in all later cell lineages. Gaining correct tissue specificity may come at the price of high level expression, leading to mosaic excision and further complications. Sorting out all these complexities requires reporter mouse lines that can give an accurate readout of the Cre activity, by activation of a ubiquitously expressed cell marker after Cre excision. Several such lines were reported at the meeting (Paul Krimpenfort, The Netherlands Cancer Institute; Sally Camper, University of Michigan Medical School; Andras Nagy and Corinne Lobe, Mount Sinai Hospital, Toronto), none of which alone provides a perfect reporter for all tissues. However, collectively, along with other lines available from other labs, the mouse seems to be covered and investigators should choose their reporter mice depending on their tissue of interest. The Cre recombinase is also the workhorse of all attempts to remodel the mammalian genome in ES cells. Many kinds of alteration can be introduced into a gene by gene targeting, but a selectable marker must always be cointroduced, which can and often does interfere with gene expression. This effect can be turned to advantage in the generation of hypomorphic alleles (Mark Lewandoski, University of California, San Francisco; A. Nagy, Toronto). However, surrounding the selectable marker with loxP sites allows its removal by Cre and generation of the required mutation, unsullied by exogenous DNA sequences. Brian Sauer (NIH), the ‘father’ of the Cre system, reported that a green fluorescent protein (GFP)–Cre fusion protein could simplify the in vitro excision process, allowing fluorescent-activated cell sorting (FACS) of Cre-transfected cells. Over 80% of fluorescent cells were found to have excised a floxed selectable marker. Cre can also be used to make chromosomal alterations, such as large deletions and inversions (Binhai Zheng, Baylor College of Medicine), duplications and deletions by transallelic recombination during meiotic recombination (Yann Herault, University of Geneva), and interand intrachromosomal rearrangements to alter receptor specificity in lymphocytes (Klaus Rajewski, University of Cologne). Cre recombinase activity also has the potential to al3Corresponding author. E-MAIL rossant@mshri.on.ca; FAX (416) 586-8588.