Efficient formation of single-copy human artificial chromosomes

Craig W. Gambogi; Gabriel J. Birchak; Elie Mer; David M. Brown; George Yankson; Kathryn Kixmoeller; Janardan N. Gavade; Josh L. Espinoza; Prakriti Kashyap; Chris L. Dupont; Glennis A. Logsdon; Patrick Heun; John I. Glass; Ben E. Black

doi:10.1126/science.adj3566

Efficient formation of single-copy human artificial chromosomes

Craig W. Gambogi, Gabriel J. Birchak, Elie Mer, David M. Brown, George Yankson, Kathryn Kixmoeller, Janardan N. Gavade, Josh L. Espinoza, Prakriti Kashyap, Chris L. Dupont, Glennis A. Logsdon, Patrick Heun, John I. Glass, Ben E. Black^*

^*Corresponding author for this work

School of Biological Sciences

Research output: Contribution to journal › Article › peer-review

Abstract

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~125-base pair DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. We describe an approach that efficiently forms single-copy HACs. It employs a ~750-kilobase construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells.

Original language	English
Pages (from-to)	1344-1349
Number of pages	6
Journal	Science
Volume	383
Issue number	6689
DOIs	https://doi.org/10.1126/science.adj3566
Publication status	Published - 22 Mar 2024

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@article{c65793cd52274aa2a6938136ec80ec32,

title = "Efficient formation of single-copy human artificial chromosomes",

abstract = "Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through \textasciitilde{}125-base pair DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. We describe an approach that efficiently forms single-copy HACs. It employs a \textasciitilde{}750-kilobase construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells. ",

author = "Gambogi, \{Craig W.\} and Birchak, \{Gabriel J.\} and Elie Mer and Brown, \{David M.\} and George Yankson and Kathryn Kixmoeller and Gavade, \{Janardan N.\} and Espinoza, \{Josh L.\} and Prakriti Kashyap and Dupont, \{Chris L.\} and Logsdon, \{Glennis A.\} and Patrick Heun and Glass, \{John I.\} and Black, \{Ben E.\}",

note = "We thank our UPenn colleagues M. Lampson (for comments on the manuscript) and M. Gerace (for assistance with preparing reagents). We also thank E. Makeyev (King{\textquoteright}s College) for reagents.",

year = "2024",

month = mar,

day = "22",

doi = "10.1126/science.adj3566",

language = "English",

volume = "383",

pages = "1344--1349",

journal = "Science",

issn = "0036-8075",

publisher = "American Association for the Advancement of Science",

number = "6689",

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TY - JOUR

T1 - Efficient formation of single-copy human artificial chromosomes

AU - Gambogi, Craig W.

AU - Birchak, Gabriel J.

AU - Mer, Elie

AU - Brown, David M.

AU - Yankson, George

AU - Kixmoeller, Kathryn

AU - Gavade, Janardan N.

AU - Espinoza, Josh L.

AU - Kashyap, Prakriti

AU - Dupont, Chris L.

AU - Logsdon, Glennis A.

AU - Heun, Patrick

AU - Glass, John I.

AU - Black, Ben E.

N1 - We thank our UPenn colleagues M. Lampson (for comments on the manuscript) and M. Gerace (for assistance with preparing reagents). We also thank E. Makeyev (King’s College) for reagents.

PY - 2024/3/22

Y1 - 2024/3/22

N2 - Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~125-base pair DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. We describe an approach that efficiently forms single-copy HACs. It employs a ~750-kilobase construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells.

AB - Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~125-base pair DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. We describe an approach that efficiently forms single-copy HACs. It employs a ~750-kilobase construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells.

UR - https://www.ncbi.nlm.nih.gov/bioproject/PRJNA985068/

U2 - 10.1126/science.adj3566

DO - 10.1126/science.adj3566

M3 - Article

SN - 0036-8075

VL - 383

SP - 1344

EP - 1349

JO - Science

JF - Science

IS - 6689

ER -

Efficient formation of single-copy human artificial chromosomes

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