Technological challenges and milestones for writing genomes

Ostrov, Nili; Beal, Jacob; Ellis, Tom; Gordon, D. Benjamin; Karas, Bogumil J.; Lee, Henry H.; Lenaghan, Scott C.; Schloss, Jeffery A.; Stracquadanio, Giovanni; Trefzer, Axel; Bader, Joel S.; Church, George M.; Coelho, Cíntia Marques; Efcavitch, J. William; Güell, Marc; Mitchell, Leslie A.; Nielsen, Alec A. K.; Peck, Bill; Smith, Alexander C.; Stewart, C. Neal; Tekotte, Hille

doi:10.1126/science.aay0339

Cited by 60 publications

(47 citation statements)

References 14 publications

(4 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The results described here have important implications for ongoing efforts to build synthetic human chromosomes by de novo synthesis 57 , as has been done with great success for budding yeast [58][59][60] . Unlike budding yeast, which has a point centromere 61 , metazoans have regional centromeres that require establishment of a proper epigenetic environment for their function and stability [62][63][64][65][66][67] .…”

Section: Discussionmentioning

confidence: 62%

Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

Pesenti

Liskovykh

Okazaki

et al. 2020

Preprint

View full text Add to dashboard Cite

AbstractHuman Artificial Chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN) and possible gene therapy. However, their use in the latter is potentially limited because the input HAC-seeding DNA can undergo an unpredictable series of rearrangements during HAC formation. As a result, after transfection and HAC formation, each cell clone contains a HAC with a unique structure that cannot be precisely predicted from the structure of the HAC-seeding DNA. Although it has been reported that these rearrangements can happen, the timing and mechanism of their formation has yet to be described. Here we synthesized a HAC-seeding DNA with two distinct structural domains and introduced it into HT1080 cells. We characterized a number of HAC-containing clones and subclones to track DNA rearrangements during HAC establishment. We demonstrated that rearrangements can occur early during HAC formation. Subsequently, the established HAC genomic organization is stably maintained across many cell generations. Thus, early stages in HAC formation appear to at least occasionally involve a process of DNA shredding and shuffling that resembles chromothripsis, an important hallmark of many cancer types. Understanding these events during HAC formation has critical implications for future efforts aimed at synthesizing and exploiting synthetic human chromosomes.

show abstract

Section: Discussionmentioning

confidence: 62%

Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

Pesenti

Liskovykh

Okazaki

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…In policy paper published last October, GP-write members highlighted technological challenges confronting the field 7 . Most notably, these include the need for alternatives to yeast as a way to assemble large DNA fragments.…”

Section: The Next Frontiermentioning

confidence: 99%

How to build a genome

Eisenstein¹

2020

Nature

View full text Add to dashboard Cite

L eslie Mitchell had no intention of doing a postdoc. After completing her PhD at the University of Ottawa, she had planned to move to industry. But then a member of her thesis committee, geneticist Jef Boeke, invited her to join his team at Johns Hopkins University in Baltimore, Maryland. Boeke was spearheading an ambitious effort to design and build an entire yeast genome from scratch, known as the Sc2.0 project. It was a once-in-a-lifetime opportunity, and one she just couldn't refuse. "I just thought that was the coolest way to study biology and really understand it," she says. "To build it from the ground up." Eight years later, the Boeke lab (now at New York University's Langone Medical Center in New York City) and its collaborators in Europe, Asia and Australia are close to producing recoded versions of all 16 Saccharomyces cerevisiae chromosomes, as well as a 17th, artificial, 'neochromosome'. Only a handful of genomes have been synthesized so far, mostly for bacteria. Synthetic biologist Jason Chin and his colleagues at the MRC Laboratory of Molecular Biology in Cambridge, UK, have rewritten the genome of Escherichia coli 1 , and researchers at the J. Craig Venter Institute (JCVI) in La Jolla, California, have constructed a 'minimal'

show abstract

“…WGS is particularly needed for engineered yeast strains which can have complex genome features like multiple deletions 9 , multiple plasmids 10 , many insertions 11 , and SCRaMbLEd chromosomes 12,13 . Furthermore, yeast are a crucial testbed for genome-scale design 14,15 , and accurate WGS will be necessary for validating written eukaryotic genomes. Finally, engineered yeast have significant economic value as promising cell factories for the manufacture of medicines 16,17 , fuels 18,19 , materials 20,21 , and chemicals 22,23 .…”

Section: Introductionmentioning

confidence: 99%

Verification of genetic engineering in yeasts with nanopore whole genome sequencing

Collins

Keating

Jones

et al. 2020

Preprint

View full text Add to dashboard Cite

Yeast genomes can be assembled from sequencing data, but genome integrations and episomal plasmids often fail to be resolved with accuracy, completeness, and contiguity. Resolution of these features is critical for many synthetic biology applications, including strain quality control and identifying engineering in unknown samples. Here, we report an integrated workflow, named Prymetime, that uses sequencing reads from inexpensive NGS platforms, assembly and error correction software, and a list of synthetic biology parts to achieve accurate whole genome sequences of yeasts with engineering annotated. To build the workflow, we first determined which sequencing methods and software packages returned an accurate, complete, and contiguous genome of an engineered S. cerevisiae strain with two similar plasmids and an integrated pathway. We then developed a sequence feature annotation step that labels synthetic biology parts from a standard list of yeast engineering sequences or from a custom sequence list. We validated the workflow by sequencing a collection of 15 engineered yeasts built from different parent S. cerevisiae and nonconventional yeast strains. We show that each integrated pathway and episomal plasmid can be correctly assembled and annotated, even in strains that have part repeats and multiple similar plasmids. Interestingly, Prymetime was able to identify deletions and unintended integrations that were subsequently confirmed by other methods. Furthermore, the whole genomes are accurate, complete, and contiguous. To illustrate this clearly, we used a publicly available S. cerevisiae CEN.PK113 reference genome and the accompanying reads to show that a Prymetime genome assembly is equivalent to the reference using several standard metrics. Finally, we used Prymetime to resequence the nonconventional yeasts Y. lipolytica Po1f and K. phaffii CBS 7435, producing an improved genome assembly for each strain. Thus, our workflow can achieve accurate, complete, and contiguous whole genome sequences of yeast strains before and after engineering. Therefore, Prymetime enables NGS-based strain quality control through assembly and identification of engineering features.Yet, applying WGS is a challenge because of the diversity of genetic backgrounds, the variety of engineering features, and the current scale of yeast strain engineering. Myriad laboratory strains of the baker's yeast Saccharomyces cerevisiae 9, 24, 25 and nonconventional yeasts like Yarrowia lipolytica 26-28 and Komagataella phaffii (formerly Pichia pastoris) 29, 30 are used to create yeast cell factories, so there are many potential genetic backgrounds. Methods of yeast engineering leave myriad sequence features behind, including standard plasmid sets with standard expression parts 31-34 , high efficiency transformation 35-37 , homologous recombination 10, 38-40 , gene knockouts using the Cre recombinase system 41 , and genome editing using RNAguided endonucleases 7,11,42,[44][45][46] . Furthermore, the scale of yeast engineering is increasing both in the...

show abstract

Technological challenges and milestones for writing genomes

Cited by 60 publications

References 14 publications

Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

How to build a genome

Verification of genetic engineering in yeasts with nanopore whole genome sequencing

Contact Info

Product

Resources

About