Fast, multiplexable and highly efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9

Abstract

Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate genes of interest. CRISPR/Cas9-based reverse genetics is a promising method to eventually achieve this. However, such methods are sorely lacking for multi-nucleated muscle fibers, since highly efficient nuclei editing is a requisite to robustly inactive candidate genes. Here, we couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery to establish a system for highly effective somatic gene mutation in mice. Using well-characterized genes, we show that local or systemic inactivation of these genes copy the phenotype of traditional gene-knockout mouse models. Thus, this proof-of-principle study establishes a method to unravel the function of individual genes or entire signaling pathways in adult skeletal muscle fibers without the cumbersome requirement of generating knockout mice.

Single-nuclei sequencing of skeletal muscle reveals subsynaptic-specific transcripts involved in neuromuscular junction maintenance

“…Nevertheless, Chrnb1 was not re-expressed in body myonuclei (Fig. 3b), confirming acute MuSK depletion was yet to induce secondary signaling effects related to denervation, as seen with chronic MuSK depletion 40 . Musk knockout (KO) significantly downregulated a discrete set of genes, not downregulated by nerve transection or BoTX injection, including Prkar1a and Ryr3 (Fig.…”

“…S3a). To identify the upstream pathways that regulate expression of particular NMJ genes, we next injected BoTX into TA muscles or blocked agrin-Lrp4/MuSK signaling at the NMJ by using the AAVMYO-CRISPR/Cas9 method to delete Musk 40, 41 . BoTX blocks ACh release from nerve terminals and thereby blocks electrical activity in postsynaptic muscle fibers, but leaves the presynaptic nerve terminal intact 42, 43 .…”

“…To get further insights into the molecular mechanisms of NMJ maintenance, we tackled the function of Etv4, Lrtm1 and Pdzrn4 , three not-described NMJ-specific genes. We used AAVMYO-mediated overexpression and a muscle fiber-specific AAVMYO-CRISPR/Cas9 knockout system, which has been described to copy the phenotype of gene knockout mice 40 . ETV4 overexpression was sufficient to trigger a synapse-specific gene transcription signature in body myonuclei by upregulating more than half of the NMJ-myonuclei transcripts (Fig.…”

“…While overexpression studies test for sufficiency of a protein, addressing essentiality requires their removal from the organism. To see whether any of the candidates were required to maintain NMJ function and structure, we used our recently established muscle fiber-specific AAV-CRISPR/Cas9 system 40 and designed six to seven sgRNAs for each gene (Supplementary Fig. S6a).…”

Efficient and multiplexed somatic genome editing with Cas12a mice

“…The generation of somatic cells with complex genotypes could be of broad value, both in cancer modeling and beyond. Somatic genome editing with Cas9 transgenic mice has enabled rapid analysis of the in vivo effects of genetic alterations in neuroscience [44][45][46][47] , cancer 21,[48][49][50][51] , immunology 52 , and other fields 53,54 . These Cas12a transgenic mice will enable the generation of pairwise and higher-order combinations of genetic alterations to be generated in cell types of interest to map complex phenotypes to complex genotypes.…”