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Two CRISPR/Cas9-mediated methods for targeting complex insertions, deletions, or replacements in mouse

Publication . Pineault, Kyriel M.; Novoa, Ana; Lozovska, Anastasiia; Wellik, Deneen M.; Mallo, Moises

Genetically modified model organisms are valuable tools for probing gene function, dissecting complex signaling networks, studying human disease, and more. CRISPR/Cas9 technology has significantly democratized and reduced the time and cost of generating genetically modified models to the point that small gene edits are now routinely and efficiently generated in as little as two months. However, generation of larger and more sophisticated gene-modifications continues to be inefficient. Alternative ways to provide the replacement DNA sequence, method of Cas9 delivery, and tethering the template sequence to Cas9 or the guide RNA (gRNA) have all been tested in an effort to maximize homology-directed repair for precise modification of the genome. We present two CRISPR/Cas9 methods that have been used to successfully generate large and complex gene-edits in mouse. In the first method, the Cas9 enzyme is used in conjunction with two sgRNAs and a long single-stranded DNA (lssDNA) template prepared by an alternative protocol. The second method utilizes a tethering approach to couple a biotinylated, double-stranded DNA (dsDNA) template to a Cas9-streptavidin fusion protein. •Alternative method for generating long, single-stranded DNA templates for CRISPR/Cas9 editing.•Demonstration that using two sgRNAs with Cas9-streptavidin/biotinylated-dsDNA is feasible for large DNA modifications.

2019Journal article

Open access

A Tgfbr1/Snai1-dependent developmental module at the core of vertebrate axial elongation

Publication . Dias, André; Lozovska, Anastasiia; Wymeersch, Filip J; Nóvoa, Ana; Binagui-Casas, Anahi; Sobral, Daniel; Martins, Gabriel G; Wilson, Valerie; Mallo, Moises

2020Journal article

Open access

Oct4 Is a Key Regulator of Vertebrate Trunk Length Diversity

Publication . Aires, Rita; Jurberg, Arnon D.; Leal, Francisca; Nóvoa, Ana; Cohn, Martin J.; Mallo, Moisés

Vertebrates exhibit a remarkably broad variation in trunk and tail lengths. However, the evolutionary and developmental origins of this diversity remain largely unknown. Posterior Hox genes were proposed to be major players in trunk length diversification in vertebrates, but functional studies have so far failed to support this view. Here we identify the pluripotency factor Oct4 as a key regulator of trunk length in vertebrate embryos. Maintaining high Oct4 levels in axial progenitors throughout development was sufficient to extend trunk length in mouse embryos. Oct4 also shifted posterior Hox gene-expression boundaries in the extended trunks, thus providing a link between activation of these genes and the transition to tail development. Furthermore, we show that the exceptionally long trunks of snakes are likely to result from heterochronic changes in Oct4 activity during body axis extension, which may have derived from differential genomic rearrangements at the Oct4 locus during vertebrate evolution.

2016-07-21Journal article

Open access

Reassessing the Role of Hox Genes during Vertebrate Development and Evolution

Publication . Mallo, Moisés

Since their discovery Hox genes have been at the core of the established models explaining the development and evolution of the vertebrate body plan as well as its paired appendages. Recent work brought new light to their role in the patterning processes along the main body axis. These studies show that Hox genes do not control the basic layout of the vertebrate body plan but carry out region-specific patterning instructions loaded on the derivatives of axial progenitors by Hox-independent processes. Furthermore, the finding that Hox clusters are embedded in functional chromatin domains, which critically impacts their expression, has significantly altered our understanding of the mechanisms of Hox gene regulation. This new conceptual framework has broadened our understanding of both limb development and the evolution of vertebrate paired appendages.

2018Journal article

Open access

Tail Bud Progenitor Activity Relies on a Network Comprising Gdf11, Lin28, and Hox13 Genes

Publication . Aires, Rita; de Lemos, Luisa; Nóvoa, Ana; Jurberg, Arnon Dias; Mascrez, Bénédicte; Duboule, Denis; Mallo, Moisés

During the trunk-to-tail transition, axial progenitors relocate from the epiblast to the tail bud. Here, we show that this process entails a major regulatory switch, bringing tail bud progenitors under Gdf11 signaling control. Gdf11 mutant embryos have an increased number of such progenitors that favor neural differentiation routes, resulting in a dramatic expansion of the neural tube. Moreover, inhibition of Gdf11 signaling recovers the proliferation ability of these progenitors when cultured in vitro. Tail bud progenitor growth is independent of Oct4, relying instead on Lin28 activity. Gdf11 signaling eventually activates Hox genes of paralog group 13, which halt expansion of these progenitors, at least in part, by down-regulating Lin28 genes. Our results uncover a genetic network involving Gdf11, Lin28, and Hox13 genes controlling axial progenitor activity in the tail bud.

2019Journal article

Open access