Biology subjects

Vigano, M. A.

Publications and source records attributed to Vigano, M. A..

3 recordsLinked to original sources

bioRxiv2022

In vivo seamless genetic engineering via CRISPR-triggered single-strand annealing

Precise genome engineering is essential for both basic and applied research. CRISPR/Cas accelerated the speed and ease by which defined exogenous sequences are integrated into specific loci. Nevertheless, knock-in generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Even when achieved, the analysis of protein localization can still be unfeasible in highly packed tissues, where spatial and temporal control of gene labeling would be ideal. Here, we describe SEED/Harvest, an efficient knock-in method based on homology-directed (HDR) and single-strand annealing (SSA) repair pathways. HDR mediates the integration of a switchable cassette. Upon a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knock-in generation with both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatio-temporal control of protein labelling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knock-ins, we have developed a toolbox based on rational engineering and functionalization of the ALFA nanobody.

genetics↗

bioRxiv2020

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

Cellular development and specialized cellular functions are regulated processes which rely on highly dynamic molecular interactions among proteins, distributed in all cell compartments. Analysis of these interactions and their mechanisms of action has been one of the main topics in cellular and developmental research over the last fifty years. Studying and understanding the functions of proteins of interest (POIs) has been mostly achieved by their alteration at the genetic level and the analysis of the phenotypic changes generated by these alterations. Although genetic and reverse genetic technologies contributed to the vast majority of information and knowledge we have gathered so far, targeting specific interactions of POIs in a time- and space-controlled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would require more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, together with several improvements in synthetic biology techniques, have contributed to the creation of a new toolbox for direct protein manipulations. We selected a number of short tag epitopes for which protein binders from different scaffolds have been developed and tested whether these tags can be bound by the corresponding protein binders in living cells when they are inserted in a single copy in a POI. We indeed find that in all cases, a single copy of a short tag allows protein binding and manipulation. Using Drosophila, we also find that single short tags can be recognized and allow degradation and relocalization of POIs in vivo.

cell biology↗

bioRxiv2018

DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo protein manipulations

Over the last few years, protein-based affinity reagents have proven very helpful in cell and developmental biology. While many of these versatile small proteins can be expressed both in the intracellular and extracellular milieu in cultured cells and in living organisms, they can also be functionalized by fusing them to different protein domains in order to regulate or modulate their target proteins in diverse manners. For example, protein binders have been employed to degrade, trap, localize or enzymatically modify specific target proteins. Whereas binders to many endogenous proteins or small protein tags have been generated, also several affinity reagents against fluorescent proteins have been created and used to manipulate target proteins tagged with the corresponding fluorescent protein. Both of these approaches have resulted in improved methods for cell biological and developmental studies. While binders against GFP and mCherry have been previously isolated and validated, we now report the generation and utilization of designed ankyrin repeat proteins (DARPins) against the monomeric teal fluorescent protein 1 (mTFP1). Here we use the generated DARPins to delocalize Rab proteins to the nuclear compartment, in which they cannot fulfill their regular functions anymore. In the future, such manipulations might enable the production of acute loss-of-function phenotypes in different cell types or living organisms based on direct protein manipulation rather than on genetic loss-of-function analyses.\n\nSummary statementStructural characterization of two novel DARPins (designed ankyrin repeat proteins) recognizing the monomeric teal fluorescent protein 1 (mTFP1) and their functionalization for protein manipulation strategies in cultured cells and potentially in living organisms.

cell biology↗