Technology used to build and transfer mammalian chromosomes

Journal Pre-proof Technology used to build and transfer mammalian chromosomes David M. Brown, John I. Glass PII:

S0014-4827(20)30045-8

DOI:

https://doi.org/10.1016/j.yexcr.2020.111851

Reference:

YEXCR 111851

To appear in:

Experimental Cell Research

Received Date: 8 November 2019 Revised Date:

9 January 2020

Accepted Date: 14 January 2020

Please cite this article as: D.M. Brown, J.I. Glass, Technology used to build and transfer mammalian chromosomes, Experimental Cell Research (2020), doi: https://doi.org/10.1016/j.yexcr.2020.111851. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.

David M. Brown: Conceptualization, Writing - Original Draft, Writing - Review & Editing, Visualization. John I. Glass: Conceptualization, Writing - Review & Editing, Funding acquisition.

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Technology used to build and transfer mammalian chromosomes

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David M. Brown1 and John I. Glass2*

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J. Craig Venter Institute, Rockville, Maryland, United States of America J. Craig Venter Institute, La Jolla, California, United States of America

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* Corresponding author

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E-mail: [email protected]

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Keywords

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Spheroplast fusion, Microcell mediated chromosome transfer (MMCT), mammalian artificial

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chromosome (MAC), human artificial chromosome (HAC), transformational-associated

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recombination (TAR) cloning

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Abstract

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In the near twenty-year existence of the human and mammalian artificial chromosome field, the

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technologies for artificial chromosome construction and installation into desired cell types or

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organisms have evolved with the rest of modern molecular and synthetic biology. Medical,

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industrial, pharmaceutical, agricultural, and basic research scientists seek the as yet unrealized

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promise of human and mammalian artificial chromosomes. Existing technologies for both top-

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down and bottom-up approaches to construct these artificial chromosomes for use in higher

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eukaryotes are very different but aspire to achieve similar results. New capacity for production

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of chromosome sized synthetic DNA will likely shift the field towards more bottom-up

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approaches, but not completely. Similarly, new approaches to install human and mammalian

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artificial chromosomes in target cells will compete with the microcell mediated cell transfer

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methods that currently dominate the field.

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Introduction

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Human and mammalian artificial chromosomes (HACs and MACs respectively) have been

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important research areas for more than twenty years. The possibility of adding new

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chromosomes comprising megabases of DNA to cells opens many opportunities. MAC/HACs

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offers advantages that other approaches to gene therapy and transgenic animal production do

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not. For instance, unlike approaches using retro and lentiviruses, when using MAC/HACs to

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genetically alter cells, the native genome of cells is unaltered. Furthermore, the size of

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MAC/HAC gene payload is much more than the 10 kb limit of most viral based gene therapy

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approaches, which makes it possible to consider adding hundreds of new genes to cells or to

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think about including entire corrected versions of damaged genes, such as the 2.4 Mb

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dystrophin gene responsible for Duchene muscular dystrophy. Unfortunately, the promise of

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MAC/HACs for gene therapies and other applications remains unfulfilled, even though

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mammalian and human artificial chromosomes have potential that could have a dramatic effect

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on healthcare.

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For a MAC/HAC to be useful in gene therapy it needs to be stable, single copy, easy to

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manipulate, and capable of carrying a payload of genetic material. Herein we summarize and

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review the technologies to generate and deliver MAC/HACs and how each technique can be

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utilized in the scheme to develop gene therapies. There have been continued advancements in

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several areas including DNA transfer and assembly technologies. These technologies can be

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linked together to create a web of different pathways for moving synthetic DNA or natural

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chromosomes to different cell types and editing them to be used for various applications. Some

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techniques are better than others and some need more refining, however manipulation of

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synthetic chromosomes may soon enter mainstream use.

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The hope for MAC/HACs is that the field will develop as yeast artificial chromosomes (YACs)

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did in the 1980s (1). The identification of the yeast centromere and autonomous replication

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sequence led to a revolution of genetic tools and capabilities in yeast. That has had a dramatic

68

effect in the field of synthetic biology and led to a catalytic expansion in our understanding of

69

yeast biology, as well as novel forms of protein expression. Building on the success of YACs,

70

yeast now has advanced genomic tools that can be utilized in the construction of other synthetic

71

chromosomes. Particularly, yeast can be used to generate HACs using a bottom-up technique

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that uses synthetic DNA to assemble a HAC. First, synthetic DNA can be constructed in vitro

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and then either transfected into a mammalian cell culture or transformed into yeast to produce

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larger, more refined constructs. The DNA can then be transferred to a mammalian cell via yeast

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fusion or by collecting the DNA from yeast and transfecting to the mammalian cells. Once

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present in a recipient cell, the HAC containing cell can be transferred or reprogramed to the cell

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type needed. The aforementioned bottom-up techniques describe one of two general

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approaches to HAC generation. The second being “top-down:” in this approach, natural HACs

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are whittled down to the functional components and then payload DNA is added to the HAC.

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This technique mainly relies on microcell mediated chromosome transfer (MMCT) to first edit

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natural chromosomes in capable cell lines and then transfer to the cell line of choice.

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In vitro DNA synthesis and engineering

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The emerging field of synthetic biology has resulted from a dramatic and rapid drop in the costs

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of DNA synthesis, which has reinvigorated the effort to generate useful HACs. Researchers can

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now order small custom genes for a few hundred dollars and receive them in just a few weeks

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(2, 3). The synthesis of DNA has become a commodity with numerous vendors capable of

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producing highly complex sequences that were impossible to synthesize just 15 years ago.

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Construction of megabase sized chromosomes has been demonstrated (4, 5).

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Robust methods of DNA synthesis and assembly can now be utilized in HAC assembly or as

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part of HAC editing. The development of approaches for synthesis of longer oligonucleotides

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containing fewer errors has resulted in DNA foundries synthesizing larger and larger pieces of

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DNA for rapid use. Contracting DNA foundries to synthesize DNA payloads is not the only

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option. Competent molecular biology labs can readily synthesize 1-3 kb DNA segments from

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oligos (6), and then assemble those segments into larger molecules. Many methods to

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assemble oligos or larger DNA fragments rely on “homology-based assembly.” One method

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referred to as Gibson Assembly (4-7) uses the T5 exonuclease to chew-back so that

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homologous ends can then anneal to the overlap regions. Other enzymes then join and repair

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the fragments.

Numerous other homology based methods have also been developed (8),

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including overlapping PCR (9), CPEC (10), SLIC (11), SLiCE (12),SIRA (13), USER (14), as

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well as many commercial kits available, including In-Fusion (Clontech), Gateway and GeneArt

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(Life Technologies), and NEBuilder® HiFi DNA Assembly (NEB).

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assembly based on Restriction digestion/ligation assembly (15) such as Biobrick (16, 17),

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golden gate (18) and LCR (19) are available. However, the trend towards cheaper commercial

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DNA synthesis means any group thinking of synthesizing HAC payloads must weigh the relative

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material and labor cost of doing it in house or contracting a foundry to make the needed DNA.

Other methods of DNA

107 108

Yeast assembly

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Brewer’s yeast, Saccharomyces cerevisiae, is an organism with extensive genetic tools thanks

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to its recombination capacity (1, 20) and, as already discussed, modern yeast genetics enable

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manipulation and construction of large DNA molecules as linear YACs or as circular yeast

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centromeric plasmids (YCPs). This means that yeast is an excellent organism in which to

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construct and then to deliver those molecules into mammalian cells. The primary process of

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manipulating synthetic DNA in yeast is transformational-associated recombination (TAR) cloning

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(1).

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assemblies including HACs (5, 7, 20-23), and synthetic yeast chromosomes (24-33).

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technique works by utilizing the recombination capacity of yeast to assemble overlapping DNA

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molecules (Figure 1).

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Originally TAR cloning was developed for genomic studies (1), where yeast could recover

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chromosome segments by “fishing out” segments of genomic DNA as large as 300 kb, which

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could then be analyzed further and create libraries of different genes. However TAR cloning is

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now frequently used in synthetic biology applications, including producing synthetic genomes,

TAR cloning is a powerful technique that can rapidly produce large synthetic DNA The

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and can be modified for more specialized assemblies (34, 35). The design of each of the TAR

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cloning fragments would be the same as for the in vitro homology-based methods described

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above, such as Gibson Assembly.

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homologous ends to each adjacent segment are constructed.

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recombine these segments together to form larger molecules. The flexibility and robustness of

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TAR cloning has been repeatedly demonstrated. Gibson and colleagues transformed yeast with

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25 overlapping fragments between 17 and 35 kb in length (7).

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have been assembled (20, 36), and oligonucleotide assembly with as many as 38 overlapping

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oligonucleotides with only 20 base overlaps into a plasmid has also been reported (37).

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Another advantage with TAR cloning compared to in vitro methods is a selected gene can be

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used to “fish out” a gene of interest from genomic DNA (1), meaning the homologous sections of

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DNA do not need to be strictly at the 5’ or 3’ ends but will recombine and assemble across the

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homologous regions to form an assembly, although with a considerable drop in efficiency.

Synthetic DNA or PCR amplified DNA containing Yeast spontaneously will

High G/C content DNA YACs

136 137

Additionally, TAR cloning can be used to build de novo HACs produced in yeast using a bottom-

138

up strategy (38). Ebersole et al. transfected yeast synthetic human alpha satellite (alphoid)

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DNA tandem repeats to produce long tandem arrays of up to 120 kb that in mammalian cells,

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associated with centromere proteins like CENP-A, form competent HACs (39). This technique

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has been used to generate a HAC with a conditional centromere, (40, 41) and is described in

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this review later as the alphoidtetO-HAC. In one example of combining the capabilities of TAR

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cloning, a portable BRCA1-HAC module for analysis of BRCA1 tumor suppressor function has

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been produced (42). Production of the BRCA1-HAC first involved the recovery of the BRCA1

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gene into a YAC and then insertion into a HAC.

146 147

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Construction of mammalian chromosomes

149

Knowledge about centromere structure, formation and stability is essential to design and build

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effective HACs. The understanding of centromere formation and de novo HAC formation has

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leapt forward in recent years. The structural components that assemble a centromere on

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chromatin are largely understood; however, the centromere organization and dynamics still

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need study. Excellent reviews on the subject have been published previously (43-48), but

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briefly, some of the most significant findings include: the discovery of a 171 bp repetitive

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sequence around where the centromere forms was identified and termed alpha satellite

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(alphoid) DNA (49). Identification of CENP-A, a H3 histone, that forms a centromere specific

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nucleosome with H2A, H2B and H4 and is actively replenished to maintain centromere identity

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around alphoid DNA (45), and is what defines a functional HAC (51, 52) was a critical discovery.

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CENP-B was the next major protein to be identified, and is the only protein that actually binds

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alphoid DNA, specifically, to a 17bp motif termed the CENP-B box (53). In total over 100

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centromere/kinetochore protein components have been identified. Another important protein is

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HJURP, which recruits CENP-A to the centromere and is stabilized by a number of remodeling

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factors (54-57). Several models for maintenance and organization of the centromere also exist

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(43, 58, 59).

165 166

As stated previously, there are two general methods of constructing HACs: “top-down” and

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“bottom-up”. Top-down HACs focus on engineering HACs from existing chromosome structures

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and has been reviewed previously (44, 60-63). Small chromosomes such as the human Y

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chromosome are whittled down to a more manageable size for experimentation.

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editing/winnowing down is typically performed based on telomere-directed chromosome

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truncation in the homologous recombination proficient chicken DT40 cell line (64). Editing is

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possible because of the notable advancement of inserting a loxP landing pad site into a HAC

The

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present in DT40 cells, allowing the HAC to be manipulated using Cre-loxP recombination (41,

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65). Minichromosomes have been produced from the human chromosomes X, Y, 14 and 21

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(64, 66-68).

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De novo HACs, or “bottom-up” engineered HACs, use alphoid DNA cloned in bacterial or yeast

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artificial chromosomes that are transfected into mammalian cells, which multimerize and form

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stable HACs 1-10 Mb as a result of the amplification of alphoid DNA. First generation HACs,

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such as in Harrington et al. (69), was produced from arrays of a 2.7 kb repeats from

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chromosome 17 to produce a final linear 173 kb product. Upon transfection into HT1080 cells,

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this HAC further multimerized and formed HACs 6-10 Mb. Once expanded to that size, the

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HAC stably replicated and segregated for over 6 months as determined by FISH. The principle

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of using repeated segments of alphoid DNA was deployed in other first generation HACs (70),

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and has generally remained today. The first observation that alphoid repeats are sufficient for

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CENP-A assembly in many cell types, enabled the first de novo HAC construction. However, a

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recent study by Black and colleagues demonstrated that a non-repetitive sequence (4q21) has

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been shown to form functional HACs without CENP-B boxes or alphoid DNA at low rates (71).

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Meaning the flexibility of how centromeres can form may be greater than previously thought.

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The rate of centromere formation on 4q21 can be increased and become possible on alphoid

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DNA is by a single pulse of epigenetic seeding of centromeric chromatin. In this case LacI-

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HJURP targeted to arrays of LacO repeats, leading to formation of CENP-A nucleosomes (71).

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Thus, it appears there are at least three ways to form a competent mammalian centromere.

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First, alphoid DNA alone can form HACs (52, 69). Second, seeding of CENP-A nucleosomes

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can form HACs (71) and third, a non- alphoid DNA (4q21) can lead to HAC formation (71).

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Another notable advancement in HAC technology is the development of the alphoidtetO-HAC.

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(72) In this HAC, a ~50 kb synthetic alphoid DNA array and a tetracycline operator (tetO) were

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combined to form a conditional centromere (40, 41, 73) such that when a fusion protein of the

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Tet Repressor (TetR) with a KRAB silencing domain (tTS) (74) is expressed the kinetochore

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disassociates and the HAC can be lost. This alphoidtetO-HAC was used in subsequent studies

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to determine how kinetochore formation occurs and what other proteins are involved in the

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process (75).

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Until recently, de novo HAC formation has been limited to very few human cell lines, notably

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HT1080 (76, 77). However, Ohzeki et al. (52) discovered that the H3K9 acetyl/methyl balance

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explains the cell specificity for de novo HAC formation. Specifically, that H3 methylation (H3K9

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methylation) is necessary for HP1 assembly, which is necessary for heterochromatin formation,

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and that the ratio of H3k9me3 is different dependent on cell type, with HeLa cells having a high

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ratio and HT1080 cells having a low ratio.

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centromere can significantly stabilize the HAC, thus allowing for the expansion of de novo HAC

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formation to other cell types (52).

Tethering histone acetyl transferase to the

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Delivery of synthetic DNA to mammalian cells by yeast fusion

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As construction of these bottom-up MAC/HACs and large DNA constructs becomes easier and

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more routine to make, the delivery of large segments of DNA to the mammalian nucleus

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remains a limiting step to effective gene therapy.

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cells, yeast spheroplasts (yeast whose cell walls were enzymatically removed) can be fused by

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a polyethylene glycol (PEG) mediated cellular fusion method (78). The advantage of this

To deliver DNA from yeast to mammalian

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method is that yeast can be used as a construction platform to build large DNA molecules (4,

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79, 80) that can then be delivered to mammalian cells. The technique was first developed in the

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1990s and has since been used to transfer yeast artificial chromosomes to several cell types

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including goat fibroblasts (81), mouse A9 cells (82), mouse ES cells (83, 84), human renal

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carcinoma cells (85), and human epithelial HEK293 cells (86, 87).

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excess of 500 kb has been used to create transgenic mice through spheroplast fusion with

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mouse embryonic stem cells (82).

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technique (83, 85, 88-90). However, previously we developed an improved method of fusing

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yeast spheroplasts by arresting the recipient mammalian cell in the mitotic phase (M-phase) of

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the cell cycle when the mammalian nuclear envelope is broken down, thus allowing for easier

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diffusion of the vector to where the mammalian nucleus re-forms (78). Importantly, the vector

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as yeast chromatin seems to be largely recognized by mammalian RNA polymerase and

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expressed, as evidenced by the recognition of delivered herpes simples virus (HSV-1) genomes

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and production of HSV-1 virions via yeast fusion (78). Interestingly, DNA delivery rates of

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vector sizes up to 1.1 Mb do not impede the DNA delivery rate and were delivered at similar

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rates to smaller vectors. The primary advantage of this method over lipofection and

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microinjection is its capacity to deliver larger DNA constructs.

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molecules to shear forces leads to breakage, decreasing delivery efficiency. This means that

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the use of agarose plugs is necessary to avoid DNA damage due to shear (91-93). In contrast,

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PEG-mediated fusion does not require DNA isolation and thus eliminates exposure of DNA to

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shear damage and potential breakage. Recently the 16 chromosomes of Saccharomyces

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cerevisiae were stitched together to form a single linear chromosome 11.8 Mb in size, which is

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the largest reported yeast chromosome to date (94), and the strain did not suffer significant

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growth problems. Another group also found a similar result reducing the number of yeast

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chromosomes to 2, each approximately 6 Mb (30). These experiments suggest it may be

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possible to construct HACs as YACs comprising millions or even tens of millions of basepairs.

In addition, yeast DNA in

This is conventionally a very low efficiency delivery

Exposure of large DNA

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Generally, the mechanism by which two membranes fuse involves two steps: aggregation and

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membrane merging. Two membranes will naturally aggregate via Van der Waals attraction

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without the hydration electrostatic forces created by water molecules that creates a strong

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repulsive barrier keeping the phospholipid membrane intact.

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binding sites or lowering the number of charges on the membrane leads to aggregation of

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nearby membranes (95, 96). Once membranes aggregate, the fusion mechanism still remains

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partially unknown, but one hypothesis is that the membranes will fuse when at least two regions

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with transient and sufficient bilayer disorder temporarily come into close contact (95). Meaning

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that regions of temporary membrane compositional disorder are required for successful cell

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fusion, and that membrane proteins can provide these defects. Without these defects, the outer

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membrane bilayer leaflets will merge, while the inner leaflets remain distinct and no mixing of

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the aqueous layer is observed (97).

Lowering the number of water

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PEG mediated cell fusion allows for easier installation of yeast borne HACs into mammalian

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cells: however, calcium, diacylglycerol, peptides, or high membrane curvature, can also induce

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fusion (97, 98). Saez et al. and Boni et al. showed by electron microscopy and X-ray scattering

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respectively that PEG-based fusion primarily functions by volume exclusion, resulting in an

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osmotic force that aggregates membranes together in a dehydrated region (98, 99). PEG-

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mediated cell fusion has been widely adopted for a wide range of cell types, although

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optimization is often needed for effective delivery and to avoid toxicity and cell lysis. The

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process has been shown to result in transfer of DNA to and from bacterial protoplasts to

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bacterial protoplasts (5, 100-104), from bacterial mini cells to yeast spheroplasts (105, 106),

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from bacterial protoplasts to mammalian cells (93, 107, 108), from yeast spheroplasts to

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mammalian cells (78, 82, 87, 109, 110), from yeast spheroplasts to insect cells (78), and from

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yeast spheroplasts to avian cells (78).

Additionally, very large DNA molecules have been

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transferred this way. A 1.1 Mb bacterial genome cloned in yeast was transplanted from into a

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different species bacterial cell (79, 104), and vectors in excess of 1 Mb have been transferred

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into mammalian cells from yeast spheroplasts (78, 82).

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The implications of this technique are that bottom-up HACs can be first constructed in yeast and

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then delivered to the mammalian cell without having to harvest in vitro DNA, minimizing shear

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forces and enabling the construction of larger synthetic DNA, over 1 Mb and perhaps

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significantly over 1 Mb. However, it is likely that top-down HACs are not amenable to yeast

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fusion, as the vectors are already present in mammalian cell lines, and these HACs are larger

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than the largest reported YACs (89).

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Microcell mediated chromosome transfer

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Microcell mediated chromosome transfer (MMCT) is a technique by which entire chromosomes

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are transferred from one mammalian cell to another by cellular fusion. It has been critical for

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development of HACs using top-down approaches. The technique was first developed in 1977

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(111). Traditionally, the process begins with arresting donor cells in metaphase using colcemid

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or colchicine as a microtubule elongation inhibitor and depolymerizer. During a transition period

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certain cells transition from metaphase to a pseudo G1 phase, called micronuclei (112, 113), a

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process by which a membrane forms around individual chromosomes. The process requires a

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donor cell that can form microcells, which is generally very limited. The most commonly used

293

cell lines are Chinese hamster ovary (CHO) cells, A9 cells and DT40 cells. Most other cell lines

294

die under prolonged exposure to microtubule inhibitors. Actin microfilaments are then disrupted

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with cytochalasin B and microcells are centrifuged through a Percoll gradient, which prevents

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cells from entering interphase (112). The purified microcells are then filtered through a 3 µm

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filter to select for microcells containing just one chromosome. Microcells can then be fused to

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the recipient cell through PEG-mediated fusion. After selection, desired cell hybrids can be

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identified. However, this traditional method suffered from very low efficiency (83, 85, 90).

300

alternative to the extended use of microtubule inhibitors was originally developed in 1975, by

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placing mitotic cells at 4°C followed by resuming g rowth at 37°C. An expanded number of cell

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lines, including HeLa cells, would create “mini-segrants” that included abnormal chromosome

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segregations very similar to microcells (114).

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pursued once MMCT was developed. Recently, several higher efficiency MMCT approaches

305

have been developed including: 1) replacement of colcemid (microtubule inhibitor) and

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cytochalasin B (actin destabilizer) with alternates like TN-16 + Griseofluvin and Latrunculin B,

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which resulted in a 6x higher efficiency (115); 2) using CHO cells to express envelope proteins

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derived from ecotropic or amphotropic murine leukemia viruses, which resulted in a 26x

309

increase in MMCT transfer rate (116); and finally 3) by retargeting the microcell fusion by adding

310

anti-Transferrin receptor (TfR) single chain antibodies (scFvs) to the extracellular C-terminus of

311

the measles virus hemagglutinin protein, which has been reported to improve the efficiency of

312

MMCT to human fibroblasts (117).

313

Because the most amenable donor cell lines to MMCT are murine models, transfer of

314

chromosomes to mouse cell lines has been relatively successful.

315

include the creation of A9 or CHO-microcell hybrid libraries that contain individual human

316

chromosomes along with each mouse chromosome; meaning these chimeric mouse cell

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libraries can provide a resource for the functional expression and mapping of human genes

318

(113, 115, 118, 119). Another major advancement was the transfer of a MAC to mouse

319

embryonic stem cells which generated a transchromosomic mouse as a stable animal model

320

(120).

One

However, this technique was generally not

Notable advancements

These can have numerous applications such as the generation of knock out or

321

transchromosomic murine models and humanized animal models (121).

Notably, the capacity

322

of yeast fusion to deliver synthetic DNA to CHO cells (78) means HACs cloned as YACs may be

323

“mobile” within MMCT, possibly linking the two techniques.

324 325

Other methods of chromosome transfer

326

Other methods of chromosome transfer exist; however, these are only possible using highly

327

specialized technicians and equipment. In addition, chromosomes to be transferred must be

328

prepared very gently. Normally the cells in which the chromosomes are constructed are

329

suspended in agarose plugs, then the chromosomes are freed from their cells by proteinase and

330

detergent treatment so that the desired large DNA molecules are safe from fluid shear forces in

331

tiny agarose caverns. To use the chromosomes, the agarose plugs are gently melted or

332

dissolved and the DNA is only pipetted with extreme gentleness (91-93). The most notable of

333

these other methods is needle microinjection. In this process, there is direct injection of DNA

334

into the nucleus of cells. This technique utilizes a thin glass microneedle to puncture both the

335

cell membrane as well as the nuclear envelope.

336

enough for the DNA vector to diffuse across the length of the needle into the nucleus.

337

Microinjection has been common practice since the 1980s (122). The technique has been used

338

to produce recombinant cell lines (123), transgenic animals (124), and for mitochondrial DNA

339

transfer (125).

The needle remains inside the cell long

340 341

Modifications of chromosomes

342

Once chromosomes are present in yeast they can be modified easily. Cre-LoxP technology is a

343

widely used site specific DNA recombination system originally derived from bacteriophage P1

344

(126, 127).

Cre recombinase catalyzes a recombination reaction between two loxP sites

345

requiring no other accessory factors. The loxP site comprises two 13 base inverted repeats

346

separated by an 8 base asymmetric spacer sequence for a total of 34 bases (126, 128). This

347

Cre-LoxP system has been utilized in yeast (129) and more recently has been used in the

348

construction of synthetic yeast chromosomes in the synthetic Sc2.0 project with all non-

349

essential genes flanked by loxPsym sites. Cre recombinase is then induced and rearranges the

350

genome, referred to as SCRaMbLE (25, 27), allowing for the study of genome organization

351

yeast evolution etc. This systematic process of using an enzyme to generate a site-specific

352

clean exchange of a cassette for an analogous cassette of choice is termed recombinase

353

mediated cassette exchange (RMCE). RMCE was originally based on the yeast integrase Flp

354

but has now mostly been replaced by the more active Cre recombinase (130). Since the

355

development of RMCE, there have been improvements such as a dual RMCE (130-132) and

356

cell lines that improve the efficiency of integration (133).

357

methods rely on the principles of TAR cloning include TREC (34), TREC-IN (35), and the

358

CRISPR based methods (134, 135) to edit, add and remove segments of DNA. Recent progress

359

using genome engineering in using CRISPR-Cas systems has also been made (136-140). All

360

these editing methods primarily rely on using the efficient double strand break and repair system

361

contained within yeast (141-144).

Other yeast based DNA modification

362 363

Although yeast has inherent and powerful modification capacity, modifications of mammalian

364

chromosomes in cell culture is now possible using Cre-loxP as well (64, 65, 145). The chicken

365

cell line DT40, a recombination competent cell line, has primarily been the main cell line used to

366

edit chromosomes using loxP technology (65, 66). In those studies, top-down HACs were

367

originally derived from natural chromosomes and edited in a top-down manner. These

368

chromosomes are then “mobile” because DT40 cells can be used for MMCT, so chromosomes

369

can be transferred to and from CHO cells and murine A9 cells. These edited chromosomes can

370

also be transferred into many recipient cell lines including murine ES cells which could be used

371

to generate HAC containing mice (41, 65, 116, 145-148).

372 373

Editing using CRISPR has been reported on plasmids using the human cell line HEK 293 cells

374

(149-151) and has continued to develop rapidly (152) with new and advanced methods being

375

reported constantly. Some important developments include CRISPR/Cas9-mediated genome

376

editing in nonhuman primates (153), modifications to MAC/HACs (154) within the cell lines CHO

377

and A9, connecting the technique to cell lines that are MMCT capable (147). Others have

378

reviewed other less popular editing methods such as zinc finger nucleases (ZFN), and

379

transcription activator-like effector nucleases (TALEN) for genome engineering (155) that have

380

also been applied to mammalian cell culture, and production of transgenic animals (156, 157).

381 382 383

Applications and Differentiated cells

384

As noted above, one of the greatest hopes for HACs is gene therapy applications. This could

385

include using cell-based therapies or perhaps to harvest stem cells from patients and install in

386

them a set of great single chain antibodies to protect them against new infectious diseases or

387

common cancers. Applications are limited only by imagination and limitations in HAC

388

technology. The transfer of artificial chromosomes to useful recipient cell lines is essential for

389

robust gene therapy applications. Several techniques exist; however, they are not robust for all

390

possible applications. Transfer of HACs to induced pluripotent stem cells (158-161) would be

391

very useful for gene therapies because these cells can first be produced from a patients

392

fibroblasts and then differentiated to the cell type of choice. A direct transfer of chromosomes

393

using MMCT to iPSC (158, 160, 162, 163) is also being attempted. Recently, Sinenko et al.

394

reported that the transfer of HACs to iPSC cells could be done by retro-MMCT and maintained

395

as an autonomous chromosome without affecting the pluripotent properties of the cells (162).

396

Although when speed is necessary, direct reprograming of differentiated somatic cells might be

397

of more use, because it reduces the time required by combining two reprograming steps to one.

398

For example, using lineage-specific factors to directly convert fibroblasts into neuronal cells

399

(164).

400 401

Somatic nuclear transfer to embryos (63, 165-168) can be used to clone transgenic animals or

402

to differentiate the embryos to the target cell line. This might be a useful technique to transfer a

403

MAC/HAC to a pluripotent cell type or embryo to create chimeric animals. In this process the

404

nucleus of a donor mammalian cell is removed and then implanted in enucleated oocytes of the

405

target mammal. It has primarily been used in cloning mammals (165, 167, 169). However in

406

some cases it has occasionally been used to for germline modification (170) or for transfer of

407

artificial chromosomes (171). However it is generally a difficult technique with low efficiency, 5-

408

15% (166).

409 410

Applications that involve the delivery of DNA along with functional protein could be developed

411

using the yeast fusion DNA delivery method. The delivery technology has already been used to

412

deliver active fluorescent protein with the DNA vector as a proof of concept. Additionally, when

413

the HSV-1 genome was delivered along with Ebola VP35 to the same mammalian cell there

414

was a differential in infectivity between different cell lines for HSV-1 generation (78). A yeast

415

strain engineered to contain the entire HSV-1 genome and to also express the Ebola derived

416

VP35, which blocks the host cell antiviral response (172, 173), could shut down host cell

417

interferon response and obtain viral replication in cells which HSV-1 had not replicated. Yeast

418

fused with Vero cells and HeLa cells showed differentials in HSV-1 generation levels depending

419

on whether VP35 was included or not (78).

420 421

Conclusion

422

Since

423

microchromosomes in the late 1990s (69), the field has made huge progress. Today, basic

424

MAC/HAC tools are in place and multiple methods can be utilized to construct and deliver

425

HACs, although there is need for much improvement. Figure 3 depicts a schematic of the

426

technologies summarized in this article and associated with the two basic approaches to

427

MAC/HAC construction and installation, “top-down” and “bottom-up.” Huge advances in DNA

428

synthesis have already made construction of megabase sized DNA molecules affordable to

429

many labs and eventually may make such synthetic molecules the only way to produce artificial

430

chromosomes. These advances are moving mainstream MAC/HAC research towards bottom-up

431

approaches, but for the foreseeable future, both top-down, bottom-up and hybrid approaches

432

will be in use.

MAC/HAC

research

dawned

with

the

first

generation

of

human

artificial

433 434

Where will MAC/HAC science be going? For some years now, some synthetic biologists have

435

advocated for a project to synthesize chromosomes, even genomes of a higher eukaryote,

436

possibly even a human genome (174). As noted above, DNA synthesis technology is steadily

437

improving. The Engineering Biology Research Consortium document, “A Research Roadmap

438

for the Next-Generation BioEconomy” projects huge advances in the size, the per nucleotide

439

error rates of synthetic oligonucleotides, and dramatic improvements in single and multiple

440

fragment DNA assembly such that whole chromosomes and genomes can be readily

441

produced within 10 years (175).

As predicted, advances in DNA synthesis will lead to

442

advances in MAC/HAC synthesis and likely make bottom-up chromosome construction the

443

norm. The race to rapidly and cheaply producing large synthetic DNA molecules has many

444

academic and industrial competitors, theseemerging technologies with real promise, are only

445

half of the technical advances needed to bring in an era of efficient gene therapy and

446

production of new drugs and animals using MAC/HACs. Synthetic chromosomes must be

447

installed and booted up in target cells more efficiently than can be done at present before

448

their promise can be realized. However, as we have tried to present in this review, HAC

449

installation technology lags behind DNA synthesis technology. More research to develop such

450

technology capable of installing large MAC/HACs is imperative.

451

In our view, considering these factors and the relative difficulties of MMCT and other non-yeast

452

base MAC/HAC installation technologies, it seems highly likely that YACs delivered to

453

mammalian cells using spheroplast fusion could be developed to produce stable HACs. Yeast

454

chromatin can be successfully expressed, replicated, and segregated in mammalian cells.

455

Cloning alphoid DNA in a yeast vector while including binding sites for histone

456

acetyltransferases and CENP-A or HJURP will likely form stable HACs once delivered into

457

mammalian cells. Using yeast to manipulate HACs would greatly extend the applicability of

458

HACs and may enable HACs capable of being easily constructed, modified, and delivered using

459

yeast vectors.

460

A driver for all of the anticipated MAC/HAC advances will be the potential of this technology for

461

gene therapies, construction of higher eukaryotic cells containing artificial chromosomes

462

encoding pharmaceuticals, the production of new transgenic animals, and likely, although not

463

discussed in this review, transgenic plants containing plant artificial chromosomes. We envision

464

the artificial chromosome technology presented here will evolve to become a major force in the

465

growing bioeconomy.

466

467

Acknowledgements: This work was supported by the Defense Advanced Research Projects

468

Agency [W911NF-11-2-0056]; and the J. Craig Venter Institute. We thank Lauren Perillo-Brown

469

for her diligent proofreading of this article.

470 471

Figure 1. Schematic representation of TAR cloning. In this example, two fragments (red and

472

blue) are PCR amplified with tails containing sequence overlaps to both a vector containing a

473

YAC (black) and to a gene in genomic DNA (green). Yeast can then recombine the overlaps to

474

assemble a desired molecule.

475 476

Figure 2. Schematic representation of yeast fusion to deliver synthetic DNA construct.

477

First, yeast is produced containing the synthetic DNA vector cloned as a YAC or YCp. Next the

478

yeast cell wall is removed by enzymatic digestion to create spheroplasts. PEG is used to fuse

479

the membrane bilayers of the two cell types. The YAC DNA then migrates across the cytoplasm

480

and is taken up by the mammalian nucleus and expressed. Endogenous yeast chromosomes

481

are lost because they have no machinery to drive replication or segregation.

482 483

Figure 3. Diagram of the technologies used to build and transfer MAC/HACs

484

The original MAC/HAC technology, a top-down strategy based on installation of new HAC

485

genetic payloads into existing small chromosomes, and MMCT for MAC/HAC installation is

486

shown on the left.

487

methods made possible by the emergence of synthetic biology and new tools in yeast and

488

bacterial genetics.

489

transferred to DT40 cells. The HAC is then truncated and the insertion of a LoxP site allows for

On the right, newer bottom-up MAC/HAC construction and installation

To construct a top-down HAC, first the full human chromosome is

490

modification and minimization of the HAC and the chromosome can then be transferred to other

491

cell types via MMCT.

492

assembling oligos, ordering genetic cassettes, or amplifying synthetic alphoid DNA via rolling

493

circle amplification. Modifications and assemblies can be made in vitro by homology-based

494

assembly, or by modifying and building HACs in yeast by TAR cloning. Next the bottom-up

495

HAC can be transferred to an animal cell line by transfection or yeast fusion for larger

496

constructs.

To construct a bottom-up HAC, first synthetic DNA is produced by

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