Dissecting long non-coding RNA function using in vivo synthetic biology

Prof. Dr. Stefan Mundlos

 

Precise spatiotemporal regulation of gene expression is essential for healthy embryonic development. Long non-coding RNAs (lncRNAs) contribute to the regulation of gene expression. However, our current understanding of lncRNAs largely derives from in vitro studies, with little known about their function in vivo. The lncRNA Maenli is an ideal model locus to study lncRNA function as it activates a specific target gene in early limb development (Allou et al., 2021). This enables a quantitative in vivo read-out without confounding genome-wide effects.

In collaboration with the Ibrahim Lab and the Boeke Lab (NYU), we have established an in vivo synthetic biology workflow that enables the synthesis and manipulation of large genomic regions, their precise insertion in the genome, then the generation of mouse embryos containing the synthetic landscape. Using this technology, we have re-constructed the 27kb Maenli lncRNA locus by assembling a fully synthetic version of the locus in yeast, then inserted it in its endogenous locus in Maenli knockout mouse embryonic stem cells and generated mouse embryos (Figure 1, below). Strikingly, synthetically reconstructed mouse and human Maenli each rescue the Maenli knockout limb phenotype, demonstrating the functionality of synthetically-produced lncRNA loci, as well as evolutionary conservation of the human and mouse loci.

Building on these proof-of-principle experiments, this project combines novel synthetic biology, CRISPR-engineered reporter embryos, and cutting-edge genomics to uncover the precise mechanism of Maenli lncRNA function in vivo. A PhD candidate in this project will gain expertise in in vivo synthetic biology, CRISPR-Cas9 genome editing, mouse embryonic stem cell culture, developmental biology, and both wet and dry lab workflows of genomic techniques (CUT&RUN, ChIP-seq, ATAC-seq and RNA-seq).
 

Reference

Allou, L., Balzano, S., Magg, A. et al. Non-coding deletions identify Maenli lncRNA as a limb-specific En1 regulator. Nature 592, 93–98 (2021). https://doi.org/10.1038/s41586-021-03208-9

 

For more information, have a look at the website of the Development & Disease group.