Publications by authors named "Leslie A Mitchell"

37 Publications

De novo assembly and delivery to mouse cells of a 101 kb functional human gene.

Genetics 2021 Mar 20. Epub 2021 Mar 20.

Institute for Systems Genetics, NYU Langone Health, New York, NY 10016.

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.
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http://dx.doi.org/10.1093/genetics/iyab038DOI Listing
March 2021

A versatile platform for locus-scale genome rewriting and verification.

Proc Natl Acad Sci U S A 2021 Mar;118(10)

Institute for Systems Genetics, NYU Langone Health, New York, NY 10016.

Routine rewriting of loci associated with human traits and diseases would facilitate their functional analysis. However, existing DNA integration approaches are limited in terms of scalability and portability across genomic loci and cellular contexts. We describe Big-IN, a versatile platform for targeted integration of large DNAs into mammalian cells. CRISPR/Cas9-mediated targeting of a landing pad enables subsequent recombinase-mediated delivery of variant payloads and efficient positive/negative selection for correct clones in mammalian stem cells. We demonstrate integration of constructs up to 143 kb, and an approach for one-step scarless delivery. We developed a staged pipeline combining PCR genotyping and targeted capture sequencing for economical and comprehensive verification of engineered stem cells. Our approach should enable combinatorial interrogation of genomic functional elements and systematic locus-scale analysis of genome function.
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http://dx.doi.org/10.1073/pnas.2023952118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958457PMC
March 2021

Synthetic Genomes.

Annu Rev Biochem 2020 06;89:77-101

Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University Langone Health, New York, NY 10016, USA; email:

DNA synthesis technology has progressed to the point that it is now practical to synthesize entire genomes. Quite a variety of methods have been developed, first to synthesize single genes but ultimately to massively edit or write from scratch entire genomes. Synthetic genomes can essentially be clones of native sequences, but this approach does not teach us much new biology. The ability to endow genomes with novel properties offers special promise for addressing questions not easily approachable with conventional gene-at-a-time methods. These include questions about evolution and about how genomes are fundamentally wired informationally, metabolically, and genetically. The techniques and technologies relating to how to design, build, and deliver big DNA at the genome scale are reviewed here. A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.
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http://dx.doi.org/10.1146/annurev-biochem-013118-110704DOI Listing
June 2020

Author Correction: Precise control of SCRaMbLE in synthetic haploid and diploid yeast.

Nat Commun 2019 02 14;10(1):839. Epub 2019 Feb 14.

Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.

The original version of this Article omitted a declaration from the Competing Interests statement, which should have included the following: 'J.D.B. is a founder and Director of the following: Neochromosome, Inc., the Center of Excellence for Engineering Biology, and CDI Labs, Inc. and serves on the Scientific Advisory Board of the following: Modern Meadow, Inc., Recombinetics, Inc., and Sample6, Inc.'. This has now been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-019-08474-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6376008PMC
February 2019

In vitro DNA SCRaMbLE.

Nat Commun 2018 05 22;9(1):1935. Epub 2018 May 22.

Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY, 10016, USA.

The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility is a powerful strategy for downstream engineering. Here we develop an in vitro method of DNA library construction based on structural variation to accomplish this goal. The "in vitro SCRaMbLE system" uses Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites. Using a β-carotene pathway designed for expression in yeast as an example, we demonstrate top-down and bottom-up in vitro SCRaMbLE, enabling optimization of biosynthetic pathway flux via the rearrangement of relevant transcription units. We show that our system provides a straightforward way to correlate phenotype and genotype and is potentially amenable to biochemical optimization in ways that the in vivo system cannot achieve.
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http://dx.doi.org/10.1038/s41467-018-03743-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964173PMC
May 2018

Heterozygous diploid and interspecies SCRaMbLEing.

Nat Commun 2018 05 22;9(1):1934. Epub 2018 May 22.

Department of Biochemistry Molecular Pharmacology and Institute for Systems Genetics, NYU Langone Health, New York, NY, 10016, USA.

SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution) is a genome restructuring technique that can be used in synthetic genomes such as that of Sc2.0, the synthetic yeast genome, which contains hundreds to thousands of strategically positioned loxPsym sites. SCRaMbLE has been used to induce rearrangements in yeast strains harboring one or more synthetic chromosomes, as well as plasmid DNA in vitro and in vivo. Here we describe a collection of heterozygous diploid strains produced by mating haploid semisynthetic Sc2.0 strains to haploid native parental strains. We subsequently demonstrate that such heterozygous diploid strains are more robust to the effects of SCRaMbLE than haploid semisynthetic strains, rapidly improve rationally selected phenotypes in SCRaMbLEd heterozygous diploids, and establish that multiple sets of independent genomic rearrangements are able to lead to similar phenotype enhancements. Finally, we show that heterozygous diploid SCRaMbLE can also be carried out in interspecies hybrid strains.
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http://dx.doi.org/10.1038/s41467-018-04157-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964232PMC
May 2018

Precise control of SCRaMbLE in synthetic haploid and diploid yeast.

Nat Commun 2018 05 22;9(1):1933. Epub 2018 May 22.

Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.

Compatibility between host cells and heterologous pathways is a challenge for constructing organisms with high productivity or gain of function. Designer yeast cells incorporating the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system provide a platform for generating genotype diversity. Here we construct a genetic AND gate to enable precise control of the SCRaMbLE method to generate synthetic haploid and diploid yeast with desired phenotypes. The yield of carotenoids is increased to 1.5-fold by SCRaMbLEing haploid strains and we determine that the deletion of YEL013W is responsible for the increase. Based on the SCRaMbLEing in diploid strains, we develop a strategy called Multiplex SCRaMbLE Iterative Cycling (MuSIC) to increase the production of carotenoids up to 38.8-fold through 5 iterative cycles of SCRaMbLE. This strategy is potentially a powerful tool for increasing the production of bio-based chemicals and for mining deep knowledge.
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http://dx.doi.org/10.1038/s41467-018-03084-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964104PMC
May 2018

L-SCRaMbLE as a tool for light-controlled Cre-mediated recombination in yeast.

Nat Commun 2018 05 22;9(1):1931. Epub 2018 May 22.

Department of Molecular Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476, Potsdam, Germany.

The synthetic yeast genome constructed by the International Synthetic Yeast Sc2.0 consortium adds thousands of loxPsym recombination sites to all 16 redesigned chromosomes, allowing the shuffling of Sc2.0 chromosome parts by the Cre-loxP recombination system thereby enabling genome evolution experiments. Here, we present L-SCRaMbLE, a light-controlled Cre recombinase for use in the yeast Saccharomyces cerevisiae. L-SCRaMbLE allows tight regulation of recombinase activity with up to 179-fold induction upon exposure to red light. The extent of recombination depends on induction time and concentration of the chromophore phycocyanobilin (PCB), which can be easily adjusted. The tool presented here provides improved recombination control over the previously reported estradiol-dependent SCRaMbLE induction system, mediating a larger variety of possible recombination events in SCRaMbLE-ing a reporter plasmid. Thereby, L-SCRaMbLE boosts the potential for further customization and provides a facile application for use in the S. cerevisiae genome re-engineering project Sc2.0 or in other recombination-based systems.
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http://dx.doi.org/10.1038/s41467-017-02208-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964156PMC
May 2018

Coupling Yeast Golden Gate and VEGAS for Efficient Assembly of the Violacein Pathway in Saccharomyces cerevisiae.

Methods Mol Biol 2018 ;1671:211-225

Institute for Systems Genetics, New York University Langone Medical Center, New York, NY, 10016, USA.

The ability to express non-native pathways in genetically tractable model systems is important for fields such as synthetic biology, genetics, and metabolic engineering. Here we describe a modular and hierarchical strategy to assemble multigene pathways for expression in S. cerevisiae. First, discrete promoter, coding sequence, and terminator parts are assembled in vitro into Transcription Units (TUs) flanked by adapter sequences using "yeast Golden Gate" (yGG), a type IIS restriction enzyme-dependent cloning strategy. Next, harnessing the natural capacity of S. cerevisiae for homologous recombination, TUs are assembled into pathways and expressed using the "Versatile Genetic Assembly System" (VEGAS) in yeast. Coupling transcription units constructed by yGG with VEGAS assembly is a generic and flexible workflow to achieve pathway expression in S. cerevisiae. This protocol describes assembly of a five TU pathway for yeast production of violacein, a pigment derived from Chromobacterium violaceum.
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http://dx.doi.org/10.1007/978-1-4939-7295-1_14DOI Listing
July 2018

Rapid and Efficient CRISPR/Cas9-Based Mating-Type Switching of .

G3 (Bethesda) 2018 01 4;8(1):173-183. Epub 2018 Jan 4.

Department of Biochemistry and Molecular Pharmacology NYU Langone Health, New York 10016

Rapid and highly efficient mating-type switching of enables a wide variety of genetic manipulations, such as the construction of strains, for instance, isogenic haploid pairs of both mating-types, diploids and polyploids. We used the CRISPR/Cas9 system to generate a double-strand break at the locus and, in a single cotransformation, both haploid and diploid cells were switched to the specified mating-type at ∼80% efficiency. The mating-type of strains carrying either rod or ring chromosome III were switched, including those lacking α and cryptic mating loci. Furthermore, we transplanted the synthetic yeast chromosome V to build a haploid polysynthetic chromosome strain by using this method together with an endoreduplication intercross strategy. The CRISPR/Cas9 mating-type switching method will be useful in building the complete synthetic yeast (Sc2.0) genome. Importantly, it is a generally useful method to build polyploids of a defined genotype and generally expedites strain construction, for example, in the construction of fully /α/α isogenic tetraploids.
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http://dx.doi.org/10.1534/g3.117.300347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765346PMC
January 2018

Synthetic genome engineering gets infectious.

Proc Natl Acad Sci U S A 2017 10 9;114(42):11006-11008. Epub 2017 Oct 9.

Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, United Kingdom;

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http://dx.doi.org/10.1073/pnas.1715365114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651789PMC
October 2017

Design of a synthetic yeast genome.

Science 2017 03;355(6329):1040-1044

Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.

We describe complete design of a synthetic eukaryotic genome, Sc2.0, a highly modified genome reduced in size by nearly 8%, with 1.1 megabases of the synthetic genome deleted, inserted, or altered. Sc2.0 chromosome design was implemented with BioStudio, an open-source framework developed for eukaryotic genome design, which coordinates design modifications from nucleotide to genome scales and enforces version control to systematically track edits. To achieve complete Sc2.0 genome synthesis, individual synthetic chromosomes built by Sc2.0 Consortium teams around the world will be consolidated into a single strain by "endoreduplication intercross." Chemically synthesized genomes like Sc2.0 are fully customizable and allow experimentalists to ask otherwise intractable questions about chromosome structure, function, and evolution with a bottom-up design strategy.
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http://dx.doi.org/10.1126/science.aaf4557DOI Listing
March 2017

Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond.

Science 2017 03;355(6329)

Department of Biochemistry and Molecular Pharmacology, New York University Langone School of Medicine, New York, NY 10016, USA.

We describe design, rapid assembly, and characterization of synthetic yeast Sc2.0 chromosome VI (synVI). A mitochondrial defect in the synVI strain mapped to synonymous coding changes within (), encoding an essential proteasome subunit; Sc2.0 coding changes reduced Pre4 protein accumulation by half. Completing Sc2.0 specifies consolidation of 16 synthetic chromosomes into a single strain. We investigated phenotypic, transcriptional, and proteomewide consequences of Sc2.0 chromosome consolidation in poly-synthetic strains. Another "bug" was discovered through proteomic analysis, associated with alteration of the transcription start due to transfer RNA deletion and loxPsym site insertion. Despite extensive genetic alterations across 6% of the genome, no major global changes were detected in the poly-synthetic strain "omics" analyses. This work sets the stage for completion of a designer, synthetic eukaryotic genome.
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http://dx.doi.org/10.1126/science.aaf4831DOI Listing
March 2017

Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome.

Science 2017 03;355(6329)

Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK.

Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by -mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.
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http://dx.doi.org/10.1126/science.aaf4791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5390853PMC
March 2017

Bug mapping and fitness testing of chemically synthesized chromosome X.

Science 2017 03;355(6329)

Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University (NYU) Langone Medical Center, New York City, NY 10016, USA.

Debugging a genome sequence is imperative for successfully building a synthetic genome. As part of the effort to build a designer eukaryotic genome, yeast synthetic chromosome X (synX), designed as 707,459 base pairs, was synthesized chemically. SynX exhibited good fitness under a wide variety of conditions. A highly efficient mapping strategy called pooled PCRTag mapping (PoPM), which can be generalized to any watermarked synthetic chromosome, was developed to identify genetic alterations that affect cell fitness ("bugs"). A series of bugs were corrected that included a large region bearing complex amplifications, a growth defect mapping to a recoded sequence in , and a loxPsym site affecting promoter function of PoPM is a powerful tool for synthetic yeast genome debugging and an efficient strategy for phenotype-genotype mapping.
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http://dx.doi.org/10.1126/science.aaf4706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679077PMC
March 2017

"Perfect" designer chromosome V and behavior of a ring derivative.

Science 2017 03;355(6329)

Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China.

Perfect matching of an assembled physical sequence to a specified designed sequence is crucial to verify design principles in genome synthesis. We designed and de novo synthesized 536,024-base pair chromosome synV in the "Build-A-Genome China" course. We corrected an initial isolate of synV to perfectly match the designed sequence using integrative cotransformation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated editing in 22 steps; synV strains exhibit high fitness under a variety of culture conditions, compared with that of wild-type V strains. A ring synV derivative was constructed, which is fully functional in under all conditions tested and exhibits lower spore viability during meiosis. Ring synV chromosome can extends Sc2.0 design principles and provides a model with which to study genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders.
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http://dx.doi.org/10.1126/science.aaf4704DOI Listing
March 2017

3D organization of synthetic and scrambled chromosomes.

Science 2017 03;355(6329)

Spatial Regulation of Genomes, Department of Genomes and Genetics, Institut Pasteur, Paris 75015, France.

Although the design of the synthetic yeast genome Sc2.0 is highly conservative with respect to gene content, the deletion of several classes of repeated sequences and the introduction of thousands of designer changes may affect genome organization and potentially alter cellular functions. We report here the Hi-C-determined three-dimensional (3D) conformations of Sc2.0 chromosomes. The absence of repeats leads to a smoother contact pattern and more precisely tractable chromosome conformations, and the large-scale genomic organization is globally unaffected by the presence of synthetic chromosome(s). Two exceptions are synIII, which lacks the silent mating-type cassettes, and synXII, specifically when the ribosomal DNA is moved to another chromosome. We also exploit the contact maps to detect rearrangements induced in SCRaMbLE (synthetic chromosome rearrangement and modification by -mediated evolution) strains.
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http://dx.doi.org/10.1126/science.aaf4597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679085PMC
March 2017

Engineering the ribosomal DNA in a megabase synthetic chromosome.

Science 2017 03;355(6329)

Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical Center, New York, NY 10011, USA.

We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromosome XII in SynXII was assembled using a two-step method, specified by successive megachunk integration and meiotic recombination-mediated assembly, producing a functional chromosome in Minor growth defect "bugs" detected in synXII, caused by deletion of tRNA genes, were rescued by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a synXII strain that would be identified as by standard DNA barcoding procedures.
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http://dx.doi.org/10.1126/science.aaf3981DOI Listing
March 2017

Regulation of claudin/zonula occludens-1 complexes by hetero-claudin interactions.

Nat Commun 2016 07 25;7:12276. Epub 2016 Jul 25.

Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Department of Medicine, Emory University, 205 Whitehead Building, 615 Michael Street, Atlanta, Georgia 30322, USA.

Claudins are tetraspan transmembrane tight-junction proteins that regulate epithelial barriers. In the distal airspaces of the lung, alveolar epithelial tight junctions are crucial to regulate airspace fluid. Chronic alcohol abuse weakens alveolar tight junctions, priming the lung for acute respiratory distress syndrome, a frequently lethal condition caused by airspace flooding. Here we demonstrate that in response to alcohol, increased claudin-5 paradoxically accompanies an increase in paracellular leak and rearrangement of alveolar tight junctions. Claudin-5 is necessary and sufficient to diminish alveolar epithelial barrier function by impairing the ability of claudin-18 to interact with a scaffold protein, zonula occludens 1 (ZO-1), demonstrating that one claudin affects the ability of another claudin to interact with the tight-junction scaffold. Critically, a claudin-5 peptide mimetic reverses the deleterious effects of alcohol on alveolar barrier function. Thus, claudin controlled claudin-scaffold protein interactions are a novel target to regulate tight-junction permeability.
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http://dx.doi.org/10.1038/ncomms12276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962485PMC
July 2016

SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes.

Genome Res 2016 Jan 13;26(1):36-49. Epub 2015 Nov 13.

High-Throughput Biology Center, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA; Department of Biomedical Engineering, School of Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA;

Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) generates combinatorial genomic diversity through rearrangements at designed recombinase sites. We applied SCRaMbLE to yeast synthetic chromosome arm synIXR (43 recombinase sites) and then used a computational pipeline to infer or unscramble the sequence of recombinations that created the observed genomes. Deep sequencing of 64 synIXR SCRaMbLE strains revealed 156 deletions, 89 inversions, 94 duplications, and 55 additional complex rearrangements; several duplications are consistent with a double rolling circle mechanism. Every SCRaMbLE strain was unique, validating the capability of SCRaMbLE to explore a diverse space of genomes. Rearrangements occurred exclusively at designed loxPsym sites, with no significant evidence for ectopic rearrangements or mutations involving synthetic regions, the 99% nonsynthetic nuclear genome, or the mitochondrial genome. Deletion frequencies identified genes required for viability or fast growth. Replacement of 3' UTR by non-UTR sequence had surprisingly little effect on fitness. SCRaMbLE generates genome diversity in designated regions, reveals fitness constraints, and should scale to simultaneous evolution of multiple synthetic chromosomes.
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http://dx.doi.org/10.1101/gr.193433.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4691749PMC
January 2016

qPCRTag Analysis--A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping.

J Vis Exp 2015 May 25(99):e52941. Epub 2015 May 25.

Department of Biochemistry and Molecular Pharmacology, Institute for Systems Genetics;

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII(1), and one synthetic chromosome arm, synIXR(2), have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.
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http://dx.doi.org/10.3791/52941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4542976PMC
May 2015

Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae.

Nucleic Acids Res 2015 Jul 8;43(13):6620-30. Epub 2015 May 8.

Department of Biochemistry and Molecular Pharmacology, New York University Langone School of Medicine, New York City, NY 10016, USA Institute for Systems Genetics, New York University Langone School of Medicine, New York City, NY 10016, USA High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

We have developed a method for assembling genetic pathways for expression in Saccharomyces cerevisiae. Our pathway assembly method, called VEGAS (Versatile genetic assembly system), exploits the native capacity of S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. In the VEGAS workflow, terminal homology between adjacent pathway genes and the assembly vector is encoded by 'VEGAS adapter' (VA) sequences, which are orthogonal in sequence with respect to the yeast genome. Prior to pathway assembly by VEGAS in S. cerevisiae, each gene is assigned an appropriate pair of VAs and assembled using a previously described technique called yeast Golden Gate (yGG). Here we describe the application of yGG specifically to building transcription units for VEGAS assembly as well as the VEGAS methodology. We demonstrate the assembly of four-, five- and six-gene pathways by VEGAS to generate S. cerevisiae cells synthesizing β-carotene and violacein. Moreover, we demonstrate the capacity of yGG coupled to VEGAS for combinatorial assembly.
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http://dx.doi.org/10.1093/nar/gkv466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513848PMC
July 2015

Yeast Golden Gate (yGG) for the Efficient Assembly of S. cerevisiae Transcription Units.

ACS Synth Biol 2015 Jul 23;4(7):853-9. Epub 2015 Mar 23.

†Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 550 First Avenue, New York, New York 10016, United States.

We have adapted the Golden Gate DNA assembly method to the assembly of transcription units (TUs) for the yeast Saccharomyces cerevisiae, in a method we call yeast Golden Gate (yGG). yGG allows for the easy assembly of TUs consisting of promoters (PRO), coding sequences (CDS), and terminators (TER). Carefully designed overhangs exposed by digestion with a type IIS restriction enzyme enable virtually seamless assembly of TUs that, in principle, contain all of the information necessary to express a gene of interest in yeast. We also describe a versatile set of yGG acceptor vectors to be used for TU assembly. These vectors can be used for low or high copy expression of assembled TUs or integration into carefully selected innocuous genomic loci. yGG provides synthetic biologists and yeast geneticists with an efficient new means by which to engineer S. cerevisiae.
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http://dx.doi.org/10.1021/sb500372zDOI Listing
July 2015

Junctional adhesion molecule A promotes epithelial tight junction assembly to augment lung barrier function.

Am J Pathol 2015 Feb 28;185(2):372-86. Epub 2014 Nov 28.

Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; Emory Alcohol and Lung Biology Center, Emory University School of Medicine, Atlanta, Georgia; Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia. Electronic address:

Epithelial barrier function is maintained by tight junction proteins that control paracellular fluid flux. Among these proteins is junctional adhesion molecule A (JAM-A), an Ig fold transmembrane protein. To assess JAM-A function in the lung, we depleted JAM-A in primary alveolar epithelial cells using shRNA. In cultured cells, loss of JAM-A caused an approximately 30% decrease in transepithelial resistance, decreased expression of the tight junction scaffold protein zonula occludens 1, and disrupted junctional localization of the structural transmembrane protein claudin-18. Consistent with findings in other organs, loss of JAM-A decreased β1 integrin expression and impaired filamentous actin formation. Using a model of mild systemic endoxotemia induced by i.p. injection of lipopolysaccharide, we report that JAM-A(-/-) mice showed increased susceptibility to pulmonary edema. On injury, the enhanced susceptibility of JAM-A(-/-) mice to edema correlated with increased, transient disruption of claudin-18, zonula occludens 1, and zonula occludens 2 localization to lung tight junctions in situ along with a delay in up-regulation of claudin-4. In contrast, wild-type mice showed no change in lung tight junction morphologic features in response to mild systemic endotoxemia. These findings support a key role of JAM-A in promoting tight junction homeostasis and lung barrier function by coordinating interactions among claudins, the tight junction scaffold, and the cytoskeleton.
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http://dx.doi.org/10.1016/j.ajpath.2014.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4305184PMC
February 2015

Circular permutation of a synthetic eukaryotic chromosome with the telomerator.

Proc Natl Acad Sci U S A 2014 Dec 5;111(48):17003-10. Epub 2014 Nov 5.

Department of Biochemistry and Molecular Pharmacology and Institute for Systems Genetics, New York University Langone School of Medicine, New York, NY 10016

Chromosome engineering is a major focus in the fields of systems biology, genetics, synthetic biology, and the functional analysis of genomes. Here, we describe the "telomerator," a new synthetic biology device for use in Saccharomyces cerevisiae. The telomerator is designed to inducibly convert circular DNA molecules into mitotically stable, linear chromosomes replete with functional telomeres in vivo. The telomerator cassette encodes convergent yeast telomere seed sequences flanking the I-SceI homing endonuclease recognition site in the center of an intron artificially transplanted into the URA3 selectable/counterselectable auxotrophic marker. We show that inducible expression of the homing endonuclease efficiently generates linear molecules, identified by using a simple plate-based screening method. To showcase its functionality and utility, we use the telomerator to circularly permute a synthetic yeast chromosome originally constructed as a circular molecule, synIXR, to generate 51 linear variants. Many of the derived linear chromosomes confer unexpected phenotypic properties. This finding indicates that the telomerator offers a new way to study the effects of gene placement on chromosomes (i.e., telomere proximity). However, that the majority of synIXR linear derivatives support viability highlights inherent tolerance of S. cerevisiae to changes in gene order and overall chromosome structure. The telomerator serves as an important tool to construct artificial linear chromosomes in yeast; the concept can be extended to other eukaryotes.
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http://dx.doi.org/10.1073/pnas.1414399111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260612PMC
December 2014

RADOM, an efficient in vivo method for assembling designed DNA fragments up to 10 kb long in Saccharomyces cerevisiae.

ACS Synth Biol 2015 Mar 4;4(3):213-20. Epub 2014 Jun 4.

†Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, 300072, PR China.

We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble ∼3 and ∼10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ∼3 kb "minichunks" from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects.
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http://dx.doi.org/10.1021/sb500241eDOI Listing
March 2015

Total synthesis of a functional designer eukaryotic chromosome.

Science 2014 04 27;344(6179):55-8. Epub 2014 Mar 27.

Department of Environmental Health Sciences, Johns Hopkins University (JHU) School of Public Health, Baltimore, MD 21205, USA.

Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871-base pair designer eukaryotic chromosome, synIII, which is based on the 316,617-base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATα allele on synIII. The complete design and synthesis of synIII establishes S. cerevisiae as the basis for designer eukaryotic genome biology.
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http://dx.doi.org/10.1126/science.1249252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4033833PMC
April 2014

Multichange isothermal mutagenesis: a new strategy for multiple site-directed mutations in plasmid DNA.

ACS Synth Biol 2013 Aug 11;2(8):473-7. Epub 2013 Mar 11.

Department of Molecular Biology and Genetics and High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Multichange ISOthermal (MISO) mutagenesis is a new technique allowing simultaneous introduction of multiple site-directed mutations into plasmid DNA by leveraging two existing ideas: QuikChange-style primers and one-step isothermal (ISO) assembly. Inversely partnering pairs of QuikChange primers results in robust, exponential amplification of linear fragments of DNA encoding mutagenic yet homologous ends. These products are amenable to ISO assembly, which efficiently assembles them into a circular, mutagenized plasmid. Because the technique relies on ISO assembly, MISO mutagenesis is additionally amenable to other relevant DNA modifications such as insertions and deletions. Here we provide a detailed description of the MISO mutagenesis concept and highlight its versatility by applying it to three experiments currently intractable with standard site-directed mutagenesis approaches. MISO mutagenesis has the potential to become widely used for site-directed mutagenesis.
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http://dx.doi.org/10.1021/sb300131wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4040258PMC
August 2013