Artificial Chromosomes in Mice Persist Over 3+ Generations

Instead of using viruses to deliver genes into cells — and dealing with all their baggage — the researchers from New York University created an artificial chromosome, about 5 million base pairs in length, that can carry dozens or hundreds of genes and can be cloned into embryos.

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I’ve written quite a lot about CRISPR and genome engineering here and elsewhere. Both are hot topics in synthetic biology, in part, because they will help treat myriad diseases. Magazines and newspapers and leading journals have all picked up on this excitement, and publish thousands of articles about CRISPR alone each year. Scientists see the hype, jump on the train, and the research area grows in a vicious feedback loop. Sometimes, though, it helps to step back and look at the end goal of gene editing for medicine: To safely alleviate cellular dysfunction. To get to that end goal, it sometimes pays to look in unexpected places. A new preprint does the trick.

It’s hard to insert and edit genes in people, and therefore it’s hard to fix certain genetic-linked conditions. Some genes have copy number variations or are wrapped in chromatin, and thus are difficult to reach. In human embryos, CRISPR “wreaks chromosomal mayhem,” according to one Nature headline, causing large DNA deletions. Perhaps more targeted editing tools, like prime or base editing, will help, but germline editing is currently off the table.

Chromosome partitioning. By Zeiss Microscopy, Flickr.

For a typical gene therapy, CRISPR machinery is packaged into a virus and injected into a mouse (or human retina). The viruses travel to target cells, enter those cells, and then insert their payload genes directly into the target cells’ genomes. Controlling where, exactly, those genes are inserted is not a solved problem. Genome integration is not ideal. Most viruses are also small, and can only carry one or a few genes at a time.

This preprint takes a different tact. Instead of using viruses to deliver genes into cells — and dealing with all their baggage — the researchers from New York University created an artificial chromosome, about 5 million base pairs in length, that can carry dozens or hundreds of genes and can be cloned into embryos.

As a proof-of-concept, a gene for green fluorescent protein was added to the artificial chromosome and was transferred into a mouse embryonic stem cell. A technique called tetraploid complementation, which combines two embryos to form a new one, was used to obtain “a single live birth of a male mouse, which presented a green-tinged eye and green fluorescent hair.” It took 13 separate attempts to get this method to work. Most mice died prematurely.

This artificial-chromosome-carrying male mouse couldn’t breed with C57Bl/6J female mice, no matter how many times he tried. But when those females were swapped out for CD-1 females — a “highly fecund” strain — the researchers got two litters of pups with 9 females and 15 males, four of which carried the artificial chromosome. This is the first instance of germline transmission for this human artificial chromosome.

But wait, there’s more. The artificial chromosome could be transmitted across three generations, through either egg or sperm. And animals with the artificial chromosome, in the third generation, bred like normal, “which suggests that the issues with breeding experienced with the founder may have been a byproduct of tetraploid complementation rather than” the artificial chromosome, the authors say. Notably: No selection pressure was required to transmit the chromosome across generations.

So why does this matter? Because nobody had ever tested this basic question. Namely, whether genes embedded on an artificial chromosome can transmit through generations of mice. Scientists have engineered bacteria with plasmids for decades because it is harder (or at least used to be harder) to insert genes directly into the genome. It’s far easier to add genes onto a plasmid (or artificial chromosome) in a test tube, and then just jolt the whole chunk of DNA into cells. This — followed by propagation across generations — is now possible for mammalian cells, and that’s a big deal.

Read more at bioRxiv.

Other Papers

(↑ = recommended article, * = open access, † = review article )

Assembly, Synthesis & Sequencing

↑*Construction of a synthetic Saccharomyces cerevisiae pan-genome neo-chromosome. Kutyna DR…Borneman AR. Nature Communications. Link

Basic Research

*Genomic analysis of a synthetic reversed sequence reveals default chromatin states in yeast and mammalian cells. Camellato B…Boeke JD. bioRxiv (preprint). Link

A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization during Plasmodium liver infection. Vijayan K…Kaushansky A. Cell Chemical Biology. Link

Biomanufacturing

*Fine-tuning of coumaric acid synthesis to increase naringenin production in yeast. Mao J…Chen Y. bioRxiv (preprint). Link

Acetyl-CoA synthesis through a bicyclic carbon-fixing pathway in gas-fermenting bacteria. Wu C…Xiong W. Nature Synthesis. Link

Tunable green syngas generation from CO2 and H2O with sunlight as the only energy input. Rashid RT…Zhou B. PNAS. Link

Engineering Halomonas bluephagenesis via small regulatory RNAs. Wang L…Chen G. Metabolic Engineering. Link

Production of Plant Sesquiterpene Lactone Parthenolide in the Yeast Cell Factory. Shi Y…Yuan Y. ACS Synthetic Biology. Link

*Efficient CRISPR/Cas12a-Based Genome-Editing Toolbox for Metabolic Engineering in Methanococcus maripaludis. Bao J, de Dios Mateos E & Scheller S. ACS Synthetic Biology. Link

*Engineering substrate specificity of HAD phosphatases and multienzyme systems development for the thermodynamic-driven manufacturing sugars. Tian C…Ma Y. Nature Communications. Link

MULTI-SCULPT: Multiplex Integration via Selective, CRISPR-Mediated, Ultralong Pathway Transformation in Yeast for Plant Natural Product Synthesis. Gong FL, Han J & Li S. ACS Synthetic Biology. Link

Photosynthetic biohybrid coculture for tandem and tunable CO2 and N2 fixation. Cestellos-Blanco S…Yang P. PNAS. Link

Biosensors

↑*Rapid biosensor development using plant hormone receptors as reprogrammable scaffolds. Beltrán J…Whitehead TA. Nature Biotechnology. Link

*A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation. Payá-Tormo L…Rubio LM. Scientific Reports. Link

*Biosensor optimization using a FRET pair based on mScarlet red fluorescent protein and an mScarlet-derived green fluorescent protein. Gohil K…Campbell RE. bioRxiv (preprint). Link

Circuits

*Model-Based Investigation of the Relationship between Regulation Level and Pulse Property of I1-FFL Gene Circuits. Ryan J…Tang X. ACS Synthetic Biology. Link

*Optimization for Predictive Gene Circuit Design Automation. Starkey F & Menolascina F. bioRxiv (preprint). Link

Model-Based Design of a Synthetic Oscillator Based on an Epigenetic Methylation Memory System. Klingel V…Radde NE. ACS Synthetic Biology. Link

Computational Tools & Models

*Control-Based Continuation: A New Approach to Prototype Synthetic Gene Networks. de Cesare I…Marucci L. ACS Synthetic Biology. Link

↑*Recurrent neural networks enable design of multifunctional synthetic human gut microbiome dynamics. Baranwal M…Venturelli OS. eLife. Link

CRISPR & Genetic Control

*Peptide fusion improves prime editing efficiency. Velimirovic M…Sherwood RI. Nature Communications. Link

↑*Magnetothermal control of temperature-sensitive repressors in superparamagnetic iron nanoparticle-coated Bacillus subtilis. Greeson EM…Contag CH. bioRxiv (preprint). Link

↑*Precise Transcript Targeting by CRISPR-Csm Complexes. Colognori D, Trinidad M & Doudna JA. bioRxiv (preprint). Link

*Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing. Lainšček D…Jerala R. Nature Communications. Link

*Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold. Liu Z, Robson P & Cheng A. bioRxiv (preprint). Link

Genome Editing

↑*Episomal editing of synthetic constructs in yeast using CRISPR. Zhao Y…Boeke JD. bioRxiv (preprint). Link

*Rapid, scalable, combinatorial genome engineering by Marker-less Enrichment and Recombination of Genetically Engineered loci (MERGE). Abdullah M…Kachroo AH. bioRxiv (preprint). Link

Medicine & Diagnostics

↑Genetically Modified Porcine-to-Human Cardiac Xenotransplantation. Griffith BP…Mohiuddin MM. The New England Journal of Medicine. Link

*Human organ rejuvenation by VEGF-A: Lessons from the skin. Keren A…Gilhar A. Science Advances. Link

*An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo. Ikemura N…Hoshino A. Science Translational Medicine. Link

Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics. Hu M…Zhou X. PNAS. Link

*Rapid and sensitive on-site genetic diagnostics of pest fruit flies using CRISPR-Cas12a. Alon DM…Pines G. bioRxiv (preprint). Link

Plants

*Advanced pathway engineering for phototrophic putrescine production. Freudenberg RA…Kruse O. Plant Biotechnology Journal. Link

*†Centromeres: from chromosome biology to biotechnology applications and synthetic genomes in plants. Zhou J…Su H. Plant Biotechnology Journal. Link

*Enhancing the accumulation of eicosapentaenoic acid and docosahexaenoic acid in transgenic Camelina through the CRISPR-Cas9 inactivation of the competing FAE1 pathway. Han L…Napier JA. Plant Biotechnology Journal. Link

Protein & Molecular Engineering

↑*Functional DNA-based cytoskeletons for synthetic cells. Zhan P…Göpfrich K. Nature Chemistry. Link

De novo design of discrete, stable 310-helix peptide assemblies. Kumar P…Woolfson DN. Nature. Link

Stem Cells

Induction of mouse totipotent stem cells by a defined chemical cocktail. Hu Y…Ding S. Nature. Link

Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses. Ang LT…Loh KM. Cell. Link

Tools & Technology

*Machine-learning-optimized Cas12a barcoding enables the recovery of single-cell lineages and transcriptional profiles. Hughes NW…Cong L. Molecular Cell. Link

*A microfluidic optimal experimental design platform for forward design of cell-free genetic networks. van Sluijs B…Huck WTS. Nature Communications. Link

*An intein-split transactivator for intersectional neural imaging and optogenetic manipulation. Chen H…Xu C. Nature Communications. Link

*Photocaged 5′ cap analogues for optical control of mRNA translation in cells. Klöcker N…Rentmeister A. Nature Chemistry. Link

*Cyclic microchip assay for measurement of hundreds of functional proteins in single neurons. Yang L…Wang J. Nature Communications. Link

Light-Induced Patterning of Electroactive Bacterial Biofilms. Zhao F…Boedicker JQ. ACS Synthetic Biology. Link

Miscellaneous

↑*Biosynthetic potential of the global ocean microbiome. Paoli L…Sunagawa S. Nature. Link

*In situ 3D bioprinting with bioconcrete bioink. Xie M…He Y. Nature Communications. Link

*Thousands of small, novel genes predicted in global phage genomes. Fremin BJ…GP-SmORF Consortium. Cell Reports. Link

*Electric-field facilitated rapid and efficient dissociation of tissues Into viable single cells. Welch EC…Tripathi A. Scientific Reports. Link

A centimeter-long bacterium with DNA contained in metabolically active, membrane-bound organelles. Volland J…Date SV. Science. Link


More soon,

— Niko

Twitter: @NikoMcCarty