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

Most 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.