Publications by authors named "Neta Agmon"

17 Publications

  • Page 1 of 1

Engineered dual selection for directed evolution of SpCas9 PAM specificity.

Nat Commun 2021 01 13;12(1):349. Epub 2021 Jan 13.

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

The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.
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http://dx.doi.org/10.1038/s41467-020-20650-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7807044PMC
January 2021

BRCA1 and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells.

Nat Struct Mol Biol 2020 02 10;27(2):179-191. Epub 2020 Feb 10.

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

Long interspersed element-1 (LINE-1, or L1) is the only autonomous retrotransposon that is active in human cells. Different host factors have been shown to influence L1 mobility; however, systematic analyses of these factors are limited. Here, we developed a high-throughput microscopy-based retrotransposition assay that identified the double-stranded break (DSB) repair and Fanconi anemia (FA) factors active in the S/G2 phase as potent inhibitors and regulators of L1 activity. In particular, BRCA1, an E3 ubiquitin ligase with a key role in several DNA repair pathways, directly affects L1 retrotransposition frequency and structure and plays a distinct role in controlling L1 ORF2 protein translation through L1 mRNA binding. These results suggest the existence of a 'battleground' at the DNA replication fork between homologous recombination (HR) factors and L1 retrotransposons and reveal a potential role for L1 in the genotypic evolution of tumors characterized by BRCA1 and HR repair deficiencies.
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http://dx.doi.org/10.1038/s41594-020-0374-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082080PMC
February 2020

Phylogenetic debugging of a complete human biosynthetic pathway transplanted into yeast.

Nucleic Acids Res 2020 01;48(1):486-499

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

Cross-species pathway transplantation enables insight into a biological process not possible through traditional approaches. We replaced the enzymes catalyzing the entire Saccharomyces cerevisiae adenine de novo biosynthesis pathway with the human pathway. While the 'humanized' yeast grew in the absence of adenine, it did so poorly. Dissection of the phenotype revealed that PPAT, the human ortholog of ADE4, showed only partial function whereas all other genes complemented fully. Suppressor analysis revealed other pathways that play a role in adenine de-novo pathway regulation. Phylogenetic analysis pointed to adaptations of enzyme regulation to endogenous metabolite level 'setpoints' in diverse organisms. Using DNA shuffling, we isolated specific amino acids combinations that stabilize the human protein in yeast. Thus, using adenine de novo biosynthesis as a proof of concept, we suggest that the engineering methods used in this study as well as the debugging strategies can be utilized to transplant metabolic pathway from any origin into yeast.
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http://dx.doi.org/10.1093/nar/gkz1098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145547PMC
January 2020

Author Correction: A scalable peptide-GPCR language for engineering multicellular communication.

Nat Commun 2019 01 29;10(1):554. Epub 2019 Jan 29.

Department of Chemistry, Columbia University, New York, NY, 10027, USA.

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/s41467-019-08545-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6351685PMC
January 2019

A scalable peptide-GPCR language for engineering multicellular communication.

Nat Commun 2018 11 29;9(1):5057. Epub 2018 Nov 29.

Department of Chemistry, Columbia University, New York, New York, 10027, USA.

Engineering multicellularity is one of the next breakthroughs for Synthetic Biology. A key bottleneck to building multicellular systems is the lack of a scalable signaling language with a large number of interfaces that can be used simultaneously. Here, we present a modular, scalable, intercellular signaling language in yeast based on fungal mating peptide/G-protein-coupled receptor (GPCR) pairs harnessed from nature. First, through genome-mining, we assemble 32 functional peptide-GPCR signaling interfaces with a range of dose-response characteristics. Next, we demonstrate that these interfaces can be combined into two-cell communication links, which serve as assembly units for higher-order communication topologies. Finally, we show 56 functional, two-cell links, which we use to assemble three- to six-member communication topologies and a three-member interdependent community. Importantly, our peptide-GPCR language is scalable and tunable by genetic encoding, requires minimal component engineering, and should be massively scalable by further application of our genome mining pipeline or directed evolution.
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http://dx.doi.org/10.1038/s41467-018-07610-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265332PMC
November 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

Low escape-rate genome safeguards with minimal molecular perturbation of .

Proc Natl Acad Sci U S A 2017 02 7;114(8):E1470-E1479. Epub 2017 Feb 7.

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

As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial- and yeast-based safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional- and recombinational-based biocontainment. We describe improving yeast -based transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible "decoy molecules" that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness.
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http://dx.doi.org/10.1073/pnas.1621250114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338387PMC
February 2017

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

Intrinsic biocontainment: multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes.

Proc Natl Acad Sci U S A 2015 Feb 26;112(6):1803-8. Epub 2015 Jan 26.

School of Medicine, The Johns Hopkins University, Baltimore, MD 21205; Institute for Systems Genetics, NYU Langone Medical Center, New York, NY 10016; and

Biocontainment may be required in a wide variety of situations such as work with pathogens, field release applications of engineered organisms, and protection of intellectual properties. Here, we describe the control of growth of the brewer's yeast, Saccharomyces cerevisiae, using both transcriptional and recombinational "safeguard" control of essential gene function. Practical biocontainment strategies dependent on the presence of small molecules require them to be active at very low concentrations, rendering them inexpensive and difficult to detect. Histone genes were controlled by an inducible promoter and controlled by 30 nM estradiol. The stability of the engineered genes was separately regulated by the expression of a site-specific recombinase. The combined frequency of generating viable derivatives when both systems were active was below detection (<10(-10)), consistent with their orthogonal nature and the individual escape frequencies of <10(-6). Evaluation of escaper mutants suggests strategies for reducing their emergence. Transcript profiling and growth test suggest high fitness of safeguarded strains, an important characteristic for wide acceptance.
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http://dx.doi.org/10.1073/pnas.1424704112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330768PMC
February 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

Effect of nuclear architecture on the efficiency of double-strand break repair.

Nat Cell Biol 2013 Jun 5;15(6):694-9. Epub 2013 May 5.

Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel.

The most dangerous insults to the genome's integrity are those that break both strands of the DNA. Double-strand breaks can be repaired by homologous recombination; in this conserved mechanism, a global genomic homology search finds sequences similar to those near the break, and uses them as a template for DNA synthesis and ligation. Chromosomes occupy restricted territories within the nucleus. We show that yeast genomic regions whose nuclear territories overlap recombine more efficiently than sequences located in spatially distant territories. Tethering of telomeres and centromeres reduces the efficiency of recombination between distant genomic loci, lowering the chances of non-allelic recombination. Our results challenge present models that posit an active scanning of the whole nuclear volume by the broken chromosomal end; they demonstrate that the search for homology is a limiting step in homologous recombination, and emphasize the importance of nuclear organization in genome maintenance.
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http://dx.doi.org/10.1038/ncb2745DOI Listing
June 2013

The role of Holliday junction resolvases in the repair of spontaneous and induced DNA damage.

Nucleic Acids Res 2011 Sep 23;39(16):7009-19. Epub 2011 May 23.

Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69979, Israel.

DNA double-strand breaks (DSBs) and other lesions occur frequently during cell growth and in meiosis. These are often repaired by homologous recombination (HR). HR may result in the formation of DNA structures called Holliday junctions (HJs), which need to be resolved to allow chromosome segregation. Whereas HJs are present in most HR events in meiosis, it has been proposed that in vegetative cells most HR events occur through intermediates lacking HJs. A recent screen in yeast has shown HJ resolution activity for a protein called Yen1, in addition to the previously known Mus81/Mms4 complex. Yeast strains deleted for both YEN1 and MMS4 show a reduction in growth rate, and are very sensitive to DNA-damaging agents. In addition, we investigate the genetic interaction of yen1 and mms4 with mutants defective in different repair pathways. We find that in the absence of Yen1 and Mms4 deletion of RAD1 or RAD52 have no further effect, whereas additional sensitivity is seen if RAD51 is deleted. Finally, we show that yeast cells are unable to carry out meiosis in the absence of both resolvases. Our results show that both Yen1 and Mms4/Mus81 play important (although not identical) roles during vegetative growth and in meiosis.
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http://dx.doi.org/10.1093/nar/gkr277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167605PMC
September 2011

Extrachromosomal circles of satellite repeats and 5S ribosomal DNA in human cells.

Mob DNA 2010 Mar 8;1(1):11. Epub 2010 Mar 8.

Department of Molecular Microbiology & Biotechnology Tel-Aviv University, Tel-Aviv 69978, Israel.

Background: Extrachomosomal circular DNA (eccDNA) is ubiquitous in eukaryotic organisms and was detected in every organism tested, including in humans. A two-dimensional gel electrophoresis facilitates the detection of eccDNA in preparations of genomic DNA. Using this technique we have previously demonstrated that most of eccDNA consists of exact multiples of chromosomal tandemly repeated DNA, including both coding genes and satellite DNA.

Results: Here we report the occurrence of eccDNA in every tested human cell line. It has heterogeneous mass ranging from less than 2 kb to over 20 kb. We describe eccDNA homologous to human alpha satellite and the SstI mega satellite. Moreover, we show, for the first time, circular multimers of the human 5S ribosomal DNA (rDNA), similar to previous findings in Drosophila and plants. We further demonstrate structures that correspond to intermediates of rolling circle replication, which emerge from the circular multimers of 5S rDNA and SstI satellite.

Conclusions: These findings, and previous reports, support the general notion that every chromosomal tandem repeat is prone to generate eccDNA in eukryoric organisms including humans. They suggest the possible involvement of eccDNA in the length variability observed in arrays of tandem repeats. The implications of eccDNA on genome biology may include mechanisms of centromere evolution, concerted evolution and homogenization of tandem repeats and genomic plasticity.
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http://dx.doi.org/10.1186/1759-8753-1-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225859PMC
March 2010

Proteasome nuclear activity affects chromosome stability by controlling the turnover of Mms22, a protein important for DNA repair.

PLoS Genet 2010 Feb 19;6(2):e1000852. Epub 2010 Feb 19.

Michael Smith Laboratories, University of British Columbia, Vancouver, Canada.

To expand the known spectrum of genes that maintain genome stability, we screened a recently released collection of temperature sensitive (Ts) yeast mutants for a chromosome instability (CIN) phenotype. Proteasome subunit genes represented a major functional group, and subsequent analysis demonstrated an evolutionarily conserved role in CIN. Analysis of individual proteasome core and lid subunit mutations showed that the CIN phenotype at semi-permissive temperature is associated with failure of subunit localization to the nucleus. The resultant proteasome dysfunction affects chromosome stability by impairing the kinetics of double strand break (DSB) repair. We show that the DNA repair protein Mms22 is required for DSB repair, and recruited to chromatin in a ubiquitin-dependent manner as a result of DNA damage. Moreover, subsequent proteasome-mediated degradation of Mms22 is necessary and sufficient for cell cycle progression through the G(2)/M arrest induced by DNA damage. Our results demonstrate for the first time that a double strand break repair protein is a proteasome target, and thus link nuclear proteasomal activity and DSB repair.
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http://dx.doi.org/10.1371/journal.pgen.1000852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824753PMC
February 2010

Analysis of repair mechanism choice during homologous recombination.

Nucleic Acids Res 2009 Aug 23;37(15):5081-92. Epub 2009 Jun 23.

Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel.

Double-strand breaks (DSBs) occur frequently during cell growth. Due to the presence of repeated sequences in the genome, repair of a single DSB can result in gene conversion, translocation, deletion or tandem duplication depending on the mechanism and the sequence chosen as partner for the recombinational repair. Here, we study how yeast cells repair a single, inducible DSB when there are several potential donors to choose from, in the same chromosome and elsewhere in the genome. We systematically investigate the parameters that affect the choice of mechanism, as well as its genetic regulation. Our results indicate that intrachromosomal homologous sequences are always preferred as donors for repair. We demonstrate the occurrence of a novel tri-partite repair product that combines ectopic gene conversion and deletion. In addition, we show that increasing the distance between two repeated sequences enhances the dependence on Rad51 for colony formation after DSB repair. This is due to a role of Rad51 in the recovery from the checkpoint signal induced by the DSB. We suggest a model for the competition between the different homologous recombination pathways. Our model explains how different repair mechanisms are able to compensate for each other during DSB repair.
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http://dx.doi.org/10.1093/nar/gkp495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731894PMC
August 2009

Evidence for rolling circle replication of tandem genes in Drosophila.

Nucleic Acids Res 2005 9;33(14):4519-26. Epub 2005 Aug 9.

Department of Molecular Microbiology and Biotechnology, Tel-Aviv University Tel Aviv 69978, Israel.

Extrachromosomal circular DNA (eccDNA) is one characteristic of the plasticity of the eukaryotic genome. It is found in various organisms and contains sequences derived primarily from repetitive chromosomal DNA. Using 2D gel electrophoresis, we have previously detected eccDNA composed of chromosomal tandem repeats throughout the life cycle of Drosophila. Here, we report for the first time evidence suggesting the occurrence of rolling circle replication of eccDNA in Drosophila. We show, on 2D gels, specific structures that can be enriched by benzoylated naphthoylated DEAE-cellulose chromatography and were identified in other systems as rolling circle intermediates (RCIs). These RCIs are homologous to histone genes, Stellate and Suppressor of Stellate, which are all organized in the chromosomes as tandem repeats. RCIs are detected throughout the life cycle of Drosophila and in cultured fly cells. These structures are found regardless of the expression of the replicated gene or of its chromosomal copy number.
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http://dx.doi.org/10.1093/nar/gki764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1184221PMC
August 2005