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

By Hannah N. Gruner, Bongmin Bae, Maebh Lynch, Daniel Oliver, Kevin So, Grant S. Mastick, Wei Yan, P. Miura

Posted 18 Feb 2019
bioRxiv DOI: 10.1101/553990

Most mammalian genes are subject to Alternative cleavage and PolyAdenylation (APA), which most commonly leads to the expression of two alternative length 3′ UTR isoforms. Long 3′ UTR variants are preferentially expressed in neuron-enriched tissues of metazoans. Their functional relevance in vivo is largely unknown. Calmodulin 1 ( Calm1) is a key integrator of calcium signaling that is required for correct neural development that generates short ( Calm1-S ) and long ( Calm1-L ) 3′ UTR mRNA isoforms via APA. We found Calm1-L expression to be largely restricted to neuronal tissues in mice with pronounced expression in the Dorsal Root Ganglion (DRG), whereas Calm1-S was more broadly expressed. Within DRG neurons, Calm1-S expression was detected in DRG axons, whereas Calm1-L was mostly restricted to soma. To study the in vivo function of Calm1-L we devised a CRISPR-Cas9 genome editing approach to generate mice that lack Calm1-L expression but maintain normal expression of Calm1-S . Three mutant mice lines were established that harbored different deletions, all of which successfully eliminated Calm1-L . Embryos lacking Calm1-L were found to have disorganized DRG axon and cell body migration. Explant DRG cultures from these mice were stimulated with Nerve Growth Factor to promote axon outgrowth and found to exhibit a dramatic increase in axon fasciculation. While overall CaM protein levels were found to be unaltered by long 3′ UTR loss, RNA stability assays in primary neurons revealed that Calm1-L is less stable than Calm1-S . Together, these results demonstrate the requirement for Calm1-L in DRG development and establish a widely applicable genome-editing strategy for generating long 3′ UTR isoform mutant mice.

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