Rapid generation of long synthetic centromeric tandem repeats for mammalian artificial chromosome formation
Inventors
Larionov, Vladimir L. • Earnshaw, William C. • Gassman, Reto • Kandels-Lewis, Stefanie • Masumoto, Hiroshi • Nakano, Megumi • Noskov, Vladimir • Kouprina, Natalay Y. • Barrett, Carl J. • Cardinale, Stefano
Assignees
University of Edinburgh • US Department of Health and Human Services
Publication Number
US-9139849-B2
Publication Date
2015-09-22
Expiration Date
2026-04-07
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Abstract
Methods are described for construction of long synthetic arrays of DNA repeats, such as alphoid repeats or other repeat sequences. The methods include concatamerization of DNA into short repeats (for instance using rolling circle amplification or directional in vitro ligation), followed by assembling the short repeats into long arrays by homologous recombination during transformation into microbe cells. These methods can be described generally as Recombinational Amplification of Repeats (RAR). The long arrays are engineered centromere-like regions that allow one to construct mammalian artificial chromosomes with a predefined centromeric region structure. Artificial chromosomes, including human artificial chromosomes with a regulated centromere, and methods of their use are also provided.
Core Innovation
Methods are provided for the rapid construction of long synthetic arrays of DNA repeats, including alphoid repeats, useful for forming mammalian artificial chromosomes (MACs) with a predefined centromeric region structure. The methods include concatamerization of DNA into short repeats using techniques such as rolling circle amplification (RCA) or directional in vitro ligation, followed by assembly of these short repeats into long arrays by homologous recombination during transformation into microbial cells, notably yeast, a process referred to as Recombinational Amplification of Repeats (RAR). The long arrays serve as engineered centromere-like regions that allow construction of artificial chromosomes, including human artificial chromosomes (HACs) with regulated centromeres.
The problem addressed arises from the difficulty in isolating, characterizing, and manipulating large alphoid tandem repeat DNA segments isolated from genomic libraries. Native alphoid arrays can span millions of base pairs and are important for centromere function, but they cannot be modified readily to elucidate structural requirements for de novo assembly of centromere structure. Current methods for HAC construction require large arrays with native or near-native sequences that are laborious to make and analyze, restricting the study of centromere function and the development of regulated artificial chromosomes.
The invention solves these issues by enabling synthesis of long, defined tandem repeats through a combination of in vitro amplification (e.g., rolling circle amplification) of repeat multimers and in vivo homologous recombination (e.g., transformation-associated recombination) in yeast, allowing both rapid generation and customized manipulation of alphoid or other DNA repeat sequences. This method allows the production of synthetic arrays from any mammalian repeat sequences or synthetic sequences at least 90% identical to such repeats, including arrays containing regulatory elements such as tet Operator (tetO) sequences that permit centromere function to be regulated conditionally. The synthetic arrays generated are competent for centromere formation and maintenance of artificial chromosomes, facilitating functional analysis, gene therapy applications, and the construction of regulatable chromosomes.
Claims Coverage
The patent includes 18 claims featuring methods of generating mammalian artificial chromosomes (MACs) with regulated centromeres containing synthetic tandem repeat sequences and tet Operator (tetO) sequences, as well as MACs produced by these methods. Main inventive features are as follows:
Method for generating a mammalian artificial chromosome with a regulated centromere
Assembling one or more engineered centromeric sequences into a vector where the engineered centromeric sequence includes a mammalian repeat sequence (alphoid or mouse minor satellite or synthetic sequence at least 90% identical thereto) and a tet Operator (tetO) sequence, wherein binding of a transactivator to the tetO sequence disrupts centromere function.
Use of homologous in vivo recombination for assembling centromeric sequences
Assembling engineered centromeric sequences via homologous in vivo recombination, particularly yeast homologous recombination (transformation-associated recombination, TAR).
Transformation-associated recombination (TAR) vector structure
Using a TAR vector comprising a yeast cassette (yeast origin of replication and yeast selectable marker), a mammalian marker sequence, and sequence containing hooks homologous to the mammalian repeat sequence, with hooks of at least 30 contiguous nucleotides having at least 90% homology to alphoid or mouse minor satellite sequences.
TAR vector further comprising bacterial replication and selection features
Extending the TAR vector to include a bacterial origin of replication and a bacterial selectable marker sequence.
Production of engineered centromeric sequences by rolling circle amplification (RCA)
Generating the engineered centromeric sequence by rolling circle amplification of a starting sequence to produce a mixture of different lengths of concatamerized repeat sequence as the RCA product.
Size ranges for RCA products and assembled centromeric regions
RCA products averaging about 1 to 5 kb, 1 to 10 kb, more than 2 kb, about 5 kb, or more than 5 kb in length; assembled centromeric regions of at least 10 kb, 20 kb, 50 kb, 70 kb, 80 kb, 100 kb or more than 100 kb in length.
Method of producing a MAC with a regulated centromere comprised of amplified repeat sequences
Selecting a starting sequence including a mammalian repeat and a tetO sequence, amplifying into tandem repeat sequences by rolling-circle amplification, and capturing the tandem repeats in a nucleic acid molecule via in vivo homologous recombination to produce a MAC competent for maintenance in mammalian cells; wherein binding of a transactivator to tetO disrupts centromere function.
MACs produced by the described methods
MACs made by the methods described above, optionally comprising an expression cassette with at least one mammalian protein encoding sequence.
The claims cover methods of constructing mammalian artificial chromosomes with regulated centromeres incorporating synthetic tandem repeats and tetO sequences, specifically via rolling circle amplification and homologous recombination using TAR vectors, as well as mammalian artificial chromosomes produced thereby, including those carrying expression cassettes.
Stated Advantages
Rapid and scalable generation of long synthetic tandem repeats with predetermined structure, overcoming the limitations of native centromeric DNA isolation and modification.
Facilitation of functional and structural analysis of centromere sequences by enabling easy nucleotide changes before amplification.
Generation of mammalian artificial chromosomes with defined centromeric regions competent for stable maintenance.
Avoidance of viral genes and proteins in artificial chromosomes, potentially reducing immunogenic responses compared to viral vectors.
Ability to incorporate large genomic loci with native regulatory elements unconstrained by size limitations.
Enables construction of regulated (conditional) centromeres, permitting experimental control over artificial chromosome stability.
Compatibility with shuttling between bacterial, yeast, and mammalian cells for easier manipulation and genetic engineering.
Documented Applications
Use in gene therapy as vectors for delivery and regulated expression of mammalian sequences.
Use in studying and characterizing native and engineered repeat structure and centromere/kinetochore function.
Functional and structural analysis of human centromeres.
Regulatable gene expression systems based on artificial chromosomes.
Pharmacology and screening for influence of drugs on aneuploidy by assessing chromosome stability and segregation.
Use in generating regulated or conditional chromosomes for experimental or therapeutic control of chromosome maintenance and loss.
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