Mätlik Kärt, Olfat Soophie, Cowlishaw Mark Cary, Moreno Eva Domenech, Ollila Saara, Andressoo Jaan-Olle
Department of Pharmacology, Faculty of Medicine & Helsinki Institute of Life Science, University of Helsinki, 00290 Helsinki, Finland.
Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society (NVS), Karolinska Institutet, 17177 Stockholm, Sweden.
Heliyon. 2023 Feb 24;9(3):e13844. doi: 10.1016/j.heliyon.2023.e13844. eCollection 2023 Mar.
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 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 allows the modulation of the expression levels of two other morphogens, and . Together, our work demonstrates the power of 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.
3'非翻译区（UTRs）通过调节mRNA稳定性和翻译来调控基因表达水平。我们之前表明，从胶质细胞系源性神经营养因子（GDNF）的3'UTR中替换负调控元件可导致内源性GDNF表达增加，同时保留其正常的时空表达模式。在此，我们开发了一种通过使用CRISPR/Cas9系统靶向3'UTR来生成超表达和低表达等位基因的方法。我们证明，在小鼠受精卵中，CRISPR/Cas9介导的从Gdnf天然3'UTR切除一段长抑制序列可增加内源性GDNF水平，在胚胎肾发育中产生与我们在GDNF组成型和条件性超表达小鼠中所描述的相似的表型改变。此外，我们表明CRISPR/Cas9介导的3'UTR靶向能够调节另外两种形态发生素的表达水平。总之，我们的工作证明了使用CRISPR/Cas9系统进行3'UTR编辑以创建超表达和低表达等位基因的能力，表明其在基因功能研究以及潜在的基因治疗中具有广泛的适用性。
