Biology subjects

Averof, M.

Publications and source records attributed to Averof, M..

3 recordsLinked to original sources

bioRxiv2018

Clonal analysis by tunable CRISPR-mediated excision

Clonal marking techniques based on the Cre/lox and Flp/FRT systems are widely used in multicellular model organisms to mark individual cells and their progeny, in order to study their morphology, growth properties and developmental fates. The same tools can be adapted to introduce specific genetic changes in a subset of cells within the body, i.e. to perform mosaic genetic analysis. Marking and manipulating distinct cell clones requires control over the frequency of clone induction, which is sometimes difficult to achieve. Here we present Valcyrie, a new method that replaces the conventional Cre or Flp recombinase-mediated excision of a marker cassette by CRISPR-mediated excision. A major advantage of this approach is that CRISPR efficiency can be tuned in a predictable fashion by manipulating the degree of sequence complementarity between the CRISPR guide RNA and its targets. We establish the method in the beetle Tribolium castaneum. We demonstrate that clone marking frequency can be tuned to generate embryos carrying single marked clones. The Valcyrie approach can be applied to a wide range of experimental settings, for example to modulate clone frequency with existing tools in established model organisms and to introduce clonal analysis in emerging experimental models.

genetics↗

bioRxiv2018

Is it possible to reconstruct an accurate cell lineage using CRISPR recorders?

Cell lineages provide the framework for understanding how multicellular organisms are built and how cell fates are decided during development. Describing cell lineages in most organisms is challenging, given the number of cells involved; even a fruit fly larva has ~50,000 cells and a small mammal has more than 1 billion cells. Recently, the idea of using CRISPR to induce mutations during development as heritable markers for lineage reconstruction has been proposed and trialled by several groups. While an attractive idea, its practical value depends on the accuracy of the cell lineages that can be generated by this method. Here, we use computer simulations to estimate the performance of this approach under different conditions. Our simulations incorporate empirical data on CRISPR-induced mutation frequencies in Drosophila. We show significant impacts from multiple biological and technical parameters - variable cell division rates, skewed mutational outcomes, target dropouts and different mutation sequencing strategies. Our approach reveals the limitations of recently published CRISPR recorders, and indicates how future implementations can be optimised to produce accurate cell lineages.

developmental biology↗

bioRxiv2017

Repeated co-option of a conserved gene regulatory module underpins the evolution of the crustacean carapace, insect wings and other flat outgrowths

Summary statementThe genes vestigial, scalloped and wingless comprise a conserved regulatory module that was co-opted repeatedly for the evolution of flat structures, such as insect wings, and crustacean carapace, tergites and coxal plates.\n\nSummaryHow novelties arise is a key question in evolutionary developmental biology. The crustacean carapace is a novelty that evolved in the early Cambrian. In an extant crustacean, Daphnia magna, the carapace grows from the body wall as a double-layered sheet with a specialized margin. We show that the growing margin of this carapace expresses vestigial, scalloped and wingless, genes that are known to play key roles in regulating growth at the insect wing margin. RNAi-mediated knockdown of scalloped and wingless impair carapace development, indicating that carapace and wing might share a common mechanism for margin outgrowth. However, carapace and wings arise in different parts of the body and their margins have different orientations, arguing that these structures have independent evolutionary origins. We show that scalloped is also expressed at the margin of unrelated flat outgrowths (tergites and coxal plates) in the distantly related crustacean Parhyale hawaiensis. Based on these observations, we propose that the vestigial-scalloped-wingless gene module has a common role in the margin of diverse flat structures, originating before the divergence of major crustacean lineages and the emergence of insects. Repeated co-option of this module occurred independently in the carapace, wing and other flat outgrowths, underpinning the evolution of distinct novelties in different arthropod lineages.

developmental biology↗