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

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.