Abstract
Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatio-temporal dynamics of gene products. We developed a multifunctional integrase (MIN) tag for rapid and versatile genome engineering that serves not only as a genetic entry site for the Bxb1 integrase but also as a novel epitope tag for standardized detection and precipitation. For the systematic study of epigenetic factors, including Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3 and Uhrf1, we generated MIN-tagged embryonic stem cell lines and created a toolbox of prefabricated modules that can be integrated via Bxb1-mediated recombination. We used these functional modules to study protein interactions and their spatio-temporal dynamics as well as gene expression and specific mutations during cellular differentiation and in response to external stimuli. Our genome engineering strategy provides a versatile open platform for efficient generation of multiple isogenic cell lines to study gene function under physiological conditions.
Highlights
In the last decades targeted gene disruption has been a widely used approach to gain first insights into gene function
The specificity of RNA guided endonucleases (RGENs) is mediated by small guide RNAs that bind to 20 bp within the target sequence and recruit the Cas9 nuclease to introduce a double strand break
Our novel genome engineering strategy relies on the CRISPR/Cas-assisted insertion of the multifunctional integrase (MIN)-tag sequence into the open reading frame of a target gene either directly
Summary
In the last decades targeted gene disruption has been a widely used approach to gain first insights into gene function. In order to gain comprehensive understanding of gene function these studies need to be complemented by more complex genetic manipulations such as fluorophore knockin, specific domain deletions or introduction of point mutations. The specificity of RGENs is mediated by small guide RNAs (gRNAs) that bind to 20 bp within the target sequence and recruit the Cas nuclease to introduce a double strand break. This two-component system has greatly facilitated the generation of gene disruptions in bacteria, plants and mammals, concerns have been raised about considerable off-target effects [5,6,7]. The low frequency of homologous recombination in mammals makes insertion of exogenous components such as fluorophore tags difficult and time-consuming
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