The Dark Matter Project

Did it work?
Hell yeah!
Read our two companion preprints to learn more:
bsky.app/profile/dark...

This recombination fuses mouse Taf1's exons 1-24 with human TAF1's exons 25-38 to form a fully functional gene that encodes mouse TAF1 protein. The recombination also removes all marker cassettes, making the final "converted" allele scarless.

The final step of engineering is what we call "conversion". It includes introducing Cre enzyme that induces recombination between the 1st loxP site in mouse Taf1 intron 24, and a 2nd loxP site located in human TAF1 intron 24.

Mouse genome rewriting and tailoring of three important disease loci - Nature This study describes a method to insert large stretches of exogenous DNA into mammalian genomes, which is used to insert human ACE2 loci into mouse to produce a model of human SARS-CoV-2 infection.

This feat of engineering was performed using a variant of a method we recently published called mSwAP-In (mammalian switching antibiotic resistance markers progressively for integration).
www.nature.com/articles/s41...

Next, we engineered mouse embryonic stem cells (mESCs), first with a loxP site in Taf1's intron 24, followed by integration of a marker cassette (a landing pad) downstream of the gene, and finally we replaced this cassette with the 78-kb human fragment.

CREEPY: CRISPR-mediated editing of synthetic episomes in yeast Abstract. Use of synthetic genomics to design and build ‘big’ DNA has revolutionized our ability to answer fundamental biological questions by employing a

Using CREEPY (CRISPR-mediated editing of synthetic episomes in yeast) we've substituted every amino acid that is different between humans and mice so that the expressed protein would be identical to mouse TAF1.
doi.org/10.1093/nar/...

But we were worried that the human TAF1 protein would be incompatible when expressed in mice (TAF1 is the largest subunit of the TFIID complex, a large protein group that is essential for initiating transcription of most genes).

We assembled this region from an XDP patient-derived BAC, using the superpower of S. cerevisiae (yeast), into a delivery-ready vector.

The pathogenic SVA insertion is in TAF1's intron 32, but we wanted to generate a partially humanized mouse model, and decided to include a 78 kb human region that spans TAF1 exons 25-38, including all intervening introns and the SVA.

Our solution? A "hybrid-convertible" allele:

This is the problem we had to tackle when we engineered the first mouse model of X-linked Dystonia Parkinsonism, a disease caused by an SVA (SINE-VNTR-Alu) retrotransposon insertion in the essential TAF1 gene.

The answer is quite simple of course: make it conditional.

But what if the lethality is induced by a 2.7 kb retrotransposon insertion into an X-linked gene? How would you CONDITIONALLY introduce this element?

How could one engineer a mouse carrying a LETHAL allele? A thread 🧵

One step closer to an LLM that understands novel DNA🧬

𝗜𝘁𝗲𝗿𝗮𝘁𝗶𝘃𝗲 𝗶𝗺𝗽𝗿𝗼𝘃𝗲𝗺𝗲𝗻𝘁 𝗼𝗳 𝗱𝗲𝗲𝗽 𝗹𝗲𝗮𝗿𝗻𝗶𝗻𝗴 𝗺𝗼𝗱𝗲𝗹𝘀 𝘂𝘀𝗶𝗻𝗴 𝘀𝘆𝗻𝘁𝗵𝗲𝘁𝗶𝗰 𝗿𝗲𝗴𝘂𝗹𝗮𝘁𝗼𝗿𝘆 𝗴𝗲𝗻𝗼𝗺𝗶𝗰𝘀.

pubmed.ncbi.nlm.nih.gov/41125441

Congrats Andre Ribeiro-Dos-Santos and Matt Maurano!

I am proud to share our work on X-linked Dystonia-Parkinsonism — a rare neurodegenerative movement disorder driven by a SINE-VNTR-Alu retrotransposon insertion in the TAF1 gene.

Read our two companion papers:
🔹 www.biorxiv.org/content/10.1...
🔹 www.biorxiv.org/content/10.1...

🧵 Highlights below!

The Dark Matter Project The Center for Synthetic Regulatory Genomics at NYU Langone Health is tasked with the study of the genome's Dark Matter - the noncoding DNA and its role in human diseases.

Read more on our website www.TheDarkMatterProject.org

Congrats to all authors, including Yu (Jeremy) Zhao, Ran Brosh, @liddelowsa.bsky.social, @jefboeke.bsky.social, Cris Bragg and others. This work was primarily funded by the Collaborative Center for XDP and NHGRI's CEGS program.

Myelin pathology is a key feature of X-linked Dystonia Parkinsonism X-linked Dystonia-Parkinsonism (XDP) is a progressive, adult-onset neurodegenerative movement disorder that predominantly affects males of Filipino descent. The disease is caused by the insertion of a...

In the 2nd preprint @pryprk.bsky.social et al. describe deep behavioral and histological analysis of XDP mice and reveal a surprising myelin pathology.
www.biorxiv.org/content/10.1...

Genome writing to dissect consequences of SVA retrotransposon disease X-Linked Dystonia Parkinsonism Human retrotransposon insertions are often associated with diseases. In the case of the neurodegenerative X-Linked Dystonia-Parkinsonism (XDP) disease, a human-specific SINE-VNTR-Alu subfamily F (SVA_...

In the 1st preprint Zhang et al. describe the engineering of the first XDP mouse model and the splicing defects induced by the presence of a pathogenic intronic SVA retrotransposon.
www.biorxiv.org/content/10.1...

We're thrilled to publish 2 exciting preprints today on the molecular pathology of X-linked Dystonia Parkinsonism!
biorxiv.org/content/10.1...
AND
biorxiv.org/content/10.1...

@nyulangone.bsky.social

Megabase-scale human genome rearrangement with programmable bridge recombinases Bridge recombinases are naturally occurring RNA-guided DNA recombinases that we previously demonstrated can programmably insert, excise, and invert DNA in vitro and in Escherichia coli. In this study,...

Megabase-scale human genome rearrangement with programmable bridge recombinases | Science www.science.org/doi/10.1126/...

Check out more news and publications on our website
www.TheDarkMatterProject.org

Structure of a polymorphic repeat at the CACNA1C schizophrenia locus | PNAS Genetic variation within intron 3 of the CACNA1C calcium channel gene is associated with schizophrenia and other neuropsychiatric disorders, but an...

Structure of a polymorphic repeat at the CACNA1C schizophrenia locus | PNAS www.pnas.org/doi/10.1073/...
Congrats to Raquel Moya and team!

The Dark Matter Project The Center for Synthetic Regulatory Genomics at NYU Langone Health is tasked with the study of the genome's Dark Matter - the noncoding DNA and its role in human diseases.

we're slow to post, but we do have a great website!
www.thedarkmatterproject.org