CRISPR/Cas9 editing reveals novel mechanisms of clustered microRNA regulation and function

Lazaros Lataniotis,Lorena Benedetti,Kathleen Steinhofel,Fatma O Kok,Anna Zampetaki,Simon M Hughes,Nathan D Lawson,Clinton A L Monfries,Andreas Albrecht,Manuel Mayr

doi:10.1038/s41598-017-09268-0

Abstract

MicroRNAs (miRNAs) are important regulators of diverse physiological and pathophysiological processes. MiRNA families and clusters are two key features in miRNA biology. Here we explore the use of CRISPR/Cas9 as a powerful tool to delineate the function and regulation of miRNA families and clusters. We focused on four miRNA clusters composed of miRNA members of the same family, homo-clusters or different families, hetero-clusters. Our results highlight different regulatory mechanisms in miRNA cluster expression. In the case of the miR-497~195 cluster, editing of miR-195 led to a significant decrease in the expression of the other miRNA in the cluster, miR-497a. Although no gene editing was detected in the miR-497a genomic locus, computational simulation revealed alteration in the three dimensional structure of the pri-miR-497~195 that may affect its processing. In cluster miR-143~145 our results imply a feed-forward regulation, although structural changes cannot be ruled out. Furthermore, in the miR-17~92 and miR-106~25 clusters no interdependency in miRNA expression was observed. Our findings suggest that CRISPR/Cas9 is a powerful gene editing tool that can uncover novel mechanisms of clustered miRNA regulation and function.

Highlights

MicroRNAs are small non coding RNAs that control gene expression posttranscriptionally[1]
The 3D remodelling indicates a clear difference between the entire structure of wt and mut[5] and to a lesser extent between wt and mut[1] with accessibility of the miR-497a stem loop being affected in both mutants. These results suggest that extensive deletions in the genomics locus of miR-195 stem loop can alter the tertiary structure of the entire miR-497~195 transcript
A large portion of miRNAs organise in miRNA clusters[5]

Summary

Introduction

MicroRNAs (miRNAs) are small non coding RNAs that control gene expression posttranscriptionally[1]. CRISPR/Cas[9] is an RNA-guided gene editing platform that is simple to design, highly specific and easy to use[12, 13]. It consists of a sole nuclease (Cas9) that identifies a conserved three nucleotide proto-adjacent motif (PAM) and cleaves both DNA strands creating a double stranded break (DSB). In the present study we explore the use of gene editing to delineate clustered miRNA regulation To this end we employed the CRISPR/Cas[9] system and determined the effect of gene editing in four miRNA clusters composed of miRNAs belonging to the same or distinct families. Our findings indicate that CRISPR/Cas[9] is a powerful technology that can provide novel insights into the structural constraints and mechanisms regulating the expression of clustered miRNAs

Methods

Results

Conclusion