In vivo modulation of endogenous gene expression via CRISPR/Cas9-mediated 3'UTR editing

Kärt Mätlik; Soophie Olfat; Mark Cary Cowlishaw; Eva Domenech Moreno; Saara Ollila; Jaan-Olle Andressoo

doi:10.1016/j.heliyon.2023.e13844

In vivo modulation of endogenous gene expression via CRISPR/Cas9-mediated 3'UTR editing

Heliyon. 2023 Feb 24;9(3):e13844. doi: 10.1016/j.heliyon.2023.e13844. eCollection 2023 Mar.

Authors

Kärt Mätlik¹, Soophie Olfat^{1

2}, Mark Cary Cowlishaw¹, Eva Domenech Moreno^{3

4}, Saara Ollila⁴, Jaan-Olle Andressoo^{1

2}

Affiliations

¹ Department of Pharmacology, Faculty of Medicine & Helsinki Institute of Life Science, University of Helsinki, 00290 Helsinki, Finland.
² Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society (NVS), Karolinska Institutet, 17177 Stockholm, Sweden.
³ Helsinki Institute of Life Science, University of Helsinki, 00290 Helsinki, Finland.
⁴ Translational Cancer Medicine Program, University of Helsinki, 00290 Helsinki, Finland.

Abstract

The 3' untranslated regions (UTRs) modulate gene expression levels by regulating mRNA stability and translation. We previously showed that the replacement of the negative regulatory elements from the 3'UTR of glial cell line-derived neurotrophic factor (GDNF) resulted in increased endogenous GDNF expression while retaining its normal spatiotemporal expression pattern. Here, we have developed a methodology for the generation of in vivo hyper- and hypomorphic alleles via 3'UTR targeting using the CRISPR/Cas9 system. We demonstrate that CRISPR/Cas9-mediated excision of a long inhibitory sequence from Gdnf native 3'UTR in mouse zygotes increases the levels of endogenous GDNF with similar phenotypic alterations in embryonic kidney development as we described in GDNF constitutive and conditional hypermorphic mice. Furthermore, we show that CRISPR/Cas9-mediated targeting of 3'UTRs in vivo allows the modulation of the expression levels of two other morphogens, Gdf11 and Bdnf. Together, our work demonstrates the power of in vivo 3'UTR editing using the CRISPR/Cas9 system to create hyper- and hypomorphic alleles, suggesting wide applicability in studies on gene function and potentially, in gene therapy.

Keywords: 3'UTR; CRISPR/Cas9; GDNF; Gene function; microRNAs.