Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by genetic mutations that lead to the disruption of dystrophin in muscle fibers. There is no curative treatment for this devastating disease. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) has emerged as a powerful tool for genetic manipulation and potential therapy. Here we demonstrate that CRIPSR-mediated genome editing efficiently excised a 23-kb genomic region on the X-chromosome covering the mutant exon 23 in a mouse model of DMD, and restored dystrophin expression and the dystrophin-glycoprotein complex at the sarcolemma of skeletal muscles in live mdx mice. Electroporation-mediated transfection of the Cas9/gRNA constructs in the skeletal muscles of mdx mice normalized the calcium sparks in response to osmotic shock. Adenovirus-mediated transduction of Cas9/gRNA greatly reduced the Evans blue dye uptake of skeletal muscles at rest and after downhill treadmill running. This study provides proof evidence for permanent gene correction in DMD.
Physical forces play important roles in regulating cell proliferation, differentiation, and death by activating intracellular signal transduction pathways. How cells sense mechanical stimulation, however, is largely unknown. Most studies focus on cellular membrane proteins such as ion channels, integrins, and receptors for growth factors as mechanosensory units. Here we show that mechanical stretch-induced c-Src protein tyrosine kinase activation is mediated through the actin filament-associated protein (AFAP). Distributed along the actin filaments, AFAP can directly active c-Src through binding to its Src homology 3 and/or 2 domains. Mutations at these specific binding sites on AFAP blocked mechanical stretch-induced c-Src activation. Therefore, mechanical force can be transmitted along the cytoskeleton, and interaction between cytoskeletal associated proteins and enzymes related to signal transduction may convert physical forces into biochemical reactions. Cytoskeleton deformation-induced proteinprotein interaction via specific binding sites may represent a novel intracellular mechanism for cells to sense mechanical stimulation.
A better understanding of the extent of convergent selection among crops could greatly improve breeding programs. We found that the quantitative trait locus
KRN2
in maize and its rice ortholog,
OsKRN2
, experienced convergent selection. These orthologs encode WD40 proteins and interact with a gene of unknown function, DUF1644, to negatively regulate grain number in both crops. Knockout of
KRN2
in maize or
OsKRN2
in rice increased grain yield by ~10% and ~8%, respectively, with no apparent trade-offs in other agronomic traits. Furthermore, genome-wide scans identified 490 pairs of orthologous genes that underwent convergent selection during maize and rice evolution, and these were enriched for two shared molecular pathways.
KRN2
, together with other convergently selected genes, provides an excellent target for future crop improvement.
Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system has emerged in recent years as a highly efficient RNA-guided gene manipulation platform. Simultaneous editing or transcriptional activation/suppression of different genes becomes feasible with the co-delivery of multiple guide RNAs (gRNAs). Here, we report that multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed from a single U6 promoter to exert genome editing of dystrophin and myosin binding protein C3 in human and mouse cells. Moreover, this strategy allows the expression of multiple gRNAs for synergistic transcription activation of follistatin when used with catalytically inactive dCas9-VP64 or dCas9-p300core fusions. Finally, the gRNAs linked by the self-cleaving ribozymes and tRNA could be expressed from RNA polymerase type II (pol II) promoters such as generic CMV and muscle/heart-specific MHCK7. This is particularly useful for in vivo applications when the packaging capacity of recombinant adeno-associated virus is limited while tissue-specific delivery of gRNAs and Cas9 is desired. Taken together, this study provides a novel strategy to enable tissue-specific expression of more than one gRNAs for multiplex gene editing from a single pol II promoter.
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