Precise removal of Calm1 long 3′ UTR isoform by CRISPR-Cas9 genome editing impairs dorsal root ganglion development in mice

Abstract

AbstractMost mammalian genes are subject to Alternative cleavage and PolyAdenylation (APA), often resulting in alternative length 3′ UTR isoforms. Thousands of extended or long 3′ UTR variants are preferentially expressed in neuron-enriched tissues of metazoans. However, the in vivo functions of these long 3′ UTR isoforms are largely unknown. Calmodulin 1 (Calm1) is a key integrator of calcium signaling that is required for correct neural development. Calm1 generates short (Calm1-S) and long 3′ UTR (Calm1-L) mRNA isoforms via APA. We found Calm1-S to be broadly expressed across mouse tissues, whereas Calm1-L expression was largely restricted to neural tissues, including the dorsal root ganglion (DRG). Using CRISPR-Cas9 genome editing, a series of mouse deletion lines were generated that successfully eliminated expression of Calm1-L while maintaining expression of Calm1-S. One of these lines, Calm1Δ3′ UTR, carried a 163 bp deletion surrounding the distal polyA site. Examination of Calm1Δ3′ UTR embryos revealed disrupted development of the DRG. In Calm1Δ3′ UTR DRG explant cultures undergoing axon outgrowth, we observed a dramatic increase in axon fasciculation. These results demonstrate a physiological role for Calm1-L in DRG development, and more generally, establish a genome-editing strategy to study in vivo functions of long 3′ UTR isoforms.Author SummaryMore than half of all human genes generate alternative mRNA isoforms which differ in the length of their 3’ Untranslated regions (3’ UTRs). Through a process called Alternative Cleavage and Polyadenylation thousands of broadly expressed genes preferentially express long 3’ UTR variants in brain tissues whereas their short 3’ UTR counterparts are more broadly expressed. A challenge to study the functions of these transcripts has been to generate loss of function mutant animals that lack a long 3’ UTR isoform but maintain expression of the corresponding short 3’ UTR isoform. Here, we used the precise, rapid, and efficient approach of CRISPR genome-editing to generate long 3’ UTR mutant mice. These mice, which do not express the long 3’ UTR of the Calmodulin 1 (Calm1) gene, exhibit impairment in the development of sensory neurons, including increased fasciculation of axons and aberrant cell body migration. This finding is important because it provides conclusive genetic evidence for a neural function of a long 3’ UTR isoform in an animal. The CRISPR genome-editing approach used here can be applied to the study of neuron-enriched long 3’ UTR isoforms, which number in the thousands and have largely unexplored functions.