Genetic switches designed for eukaryotic cells and controlled by serine integrases

Mayna S Gomide,Cristiano Lacorte,Maria L Robledo,André M Murad,Lilian H Florentino,Rayane N Lima,Eduardo O Melo,Gustavo P C José,Marco A De Oliveira,Martín H Bonamino,Cintia M Coelho,Stevens K Rehen,Leila M G Barros,Mariana S M Almeida,Thais T Sales,Elibio Rech,Luciana R C Barros,Cintia G Limia

doi:10.1038/s42003-020-0971-8

Abstract

Recently, new serine integrases have been identified, increasing the possibility of scaling up genomic modulation tools. Here, we describe the use of unidirectional genetic switches to evaluate the functionality of six serine integrases in different eukaryotic systems: the HEK 293T cell lineage, bovine fibroblasts and plant protoplasts. Moreover, integrase activity was also tested in human cell types of therapeutic interest: peripheral blood mononuclear cells (PBMCs), neural stem cells (NSCs) and undifferentiated embryonic stem (ES) cells. The switches were composed of plasmids designed to flip two different genetic parts driven by serine integrases. Cell-based assays were evaluated by measurement of EGFP fluorescence and by molecular analysis of attL/attR sites formation after integrase functionality. Our results demonstrate that all the integrases were capable of inverting the targeted DNA sequences, exhibiting distinct performances based on the cell type or the switchable genetic sequence. These results should support the development of tunable genetic circuits to regulate eukaryotic gene expression.

Highlights

New serine integrases have been identified, increasing the possibility of scaling up genomic modulation tools
We designed unidirectional genetic switches composed of two sets of synthesized plasmids
Tools based on activators and repressors, including recently modified CRISPR-Cas[9] systems[42], are used to regulate eukaryotic gene expression, there remains a need for new technologies that are scalable and precise for multiplex gene regulation control

Summary

Introduction

New serine integrases have been identified, increasing the possibility of scaling up genomic modulation tools. The phageencoded serine integrases (Ints) are capable of unidirectional recombination that leads to permanent DNA fragment inversion, which could be used to modulate gene expression[19] To perform this function, Ints recognize specific attachment sites, named attB and attP20. GFP expression for every Int used was observed as predicted by the truth table of each designed logic gate that was evaluated[22,23] Another strategy used to regulate prokaryotic gene expression was recombinase-based state machines, a system in which several combinations of integrase inputs that inverted or excised genetic parts produced different outputs[24]. In an attempt to fill this existing gap in eukaryotic cells, we built unidirectional genetic switches to evaluate the functionality of six out of these 11 Ints that have already been tested in prokaryotic cells

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