Transforming ecology and conservation biology through genome editing.

As the conservation challenges increase, new approaches are needed to help combat losses in biodiversity and slow or reverse the decline of threatened species. Genome-editing technology is changing the face of modern biology, facilitating applications that were unimaginable only a decade ago. The technology has the potential to make significant contributions to the fields of evolutionary biology, ecology, and conservation, yet the fear of unintended consequences from designer ecosystems containing engineered organisms has stifled innovation. To overcome this gap in the understanding of what genome editing is and what its capabilities are, more research is needed to translate genome-editing discoveries into tools for ecological research. Emerging and future genome-editing technologies include new clustered regularly interspaced short palindromic repeats (CRISPR) targeted sequencing and nucleic acid detection approaches as well as species genetic barcoding and somatic genome-editing technologies. These genome-editing tools have the potential to transform the environmental sciences by providing new noninvasive methods for monitoring threatened species or for enhancing critical adaptive traits. A pioneering effort by the conservation community is required to apply these technologies to real-world conservation problems.

Transformación de la Ecología y la Biología de la Conservación por medio de la Edición Genómica Resumen Conforme aumentan los retos de conservación, se necesitan nuevas estrategias para ayudar a combatir las pérdidas de biodiversidad y para disminuir o revertir la declinación de especies. La tecnología de edición genómica está cambiando el rostro de la biología moderna, facilitando aplicaciones que eran inimaginables hace una década. Esta tecnología tiene el potencial de contribuir significativamente en los campos de la biología evolutiva, la ecología y la conservación, aun así, el miedo a las consecuencias accidentales de los ecosistemas planeados que contienen organismos diseñados ha sofocado a la innovación. Para sobreponerse a este vacío en el entendimiento de lo que es la edición genómica y cuáles son sus capacidades se requiere de mayor investigación para traducir los descubrimientos de la edición genómica a herramientas para la investigación ecológica. Las tecnologías de edición genómica emergentes y futuras incluyen nuevas estrategias CRISPR enfocadas en la secuenciación y detección de ácidos nucleicos, así como tecnologías de definición del código de barras genético de las especies y de edición somática de genes. Estas herramientas de edición genómica tienen el potencial para transformar las ciencias ambientales al proporcionar nuevos métodos no invasivos para el monitoreo de especies amenazadas o para mejorar las características adaptativas más importantes. Se requiere de un esfuerzo vanguardista por parte de la comunidad conservadora para aplicar esta tecnología a los problemas de conservación en el mundo real.

CRISPR screen identifies the NCOR/HDAC3 complex as a major suppressor of differentiation in rhabdomyosarcoma.

Dysregulated gene expression resulting from abnormal epigenetic alterations including histone acetylation and deacetylation has been demonstrated to play an important role in driving tumor growth and progression. However, the mechanisms by which specific histone deacetylases (HDACs) regulate differentiation in solid tumors remains unclear. Using pediatric rhabdomyosarcoma (RMS) as a paradigm to elucidate the mechanism blocking differentiation in solid tumors, we identified HDAC3 as a major suppressor of myogenic differentiation from a high-efficiency Clustered regularly interspaced short palindromic repeats (CRISPR)-based phenotypic screen of class I and II HDAC genes. Detailed characterization of the HDAC3-knockout phenotype in vitro and in vivo using a tamoxifen-inducible CRISPR targeting strategy demonstrated that HDAC3 deacetylase activity and the formation of a functional complex with nuclear receptor corepressors (NCORs) were critical in restricting differentiation in RMS. The NCOR/HDAC3 complex specifically functions by blocking myoblast determination protein 1 (MYOD1)-mediated activation of myogenic differentiation. Interestingly, there was also a transient up-regulation of growth-promoting genes upon initial HDAC3 targeting, revealing a unique cancer-specific response to the forced transition from a neoplastic state to terminal differentiation. Our study applied modifications of CRISPR/CRISPR-associated endonuclease 9 (Cas9) technology to interrogate the function of essential cancer genes and pathways and has provided insights into cancer cell adaptation in response to altered differentiation status. Because current pan-HDAC inhibitors have shown disappointing results in clinical trials of solid tumors, therapeutic targets specific to HDAC3 function represent a promising option for differentiation therapy in malignant tumors with dysregulated HDAC3 activity.

Gene Function is a Driver of Activin Signaling Pathway Evolution Following Whole-Genome Duplication in Rainbow Trout (Oncorhynchus mykiss).

The genomes of plant and animal species are influenced by ancestral whole-genome duplication (WGD) events, which have profound impacts on the regulation and function of gene networks. To gain insight into the consequences of WGD events, we characterized the sequence conservation and expression patterns of ohnologs in the highly duplicated activin receptor signaling pathway in rainbow trout (RBT). The RBT activin receptor signaling pathway is defined by tissue-specific expression of inhibitors and ligands and broad expression of receptors and Co-Smad signaling molecules. Signaling pathway ligands exhibited shared expression, while inhibitors and Smad signaling molecules primarily express a single dominant ohnolog. Our findings suggest that gene function influences ohnolog evolution following duplication of the activin signaling pathway in RBT.

Muscle growth in teleost fish is regulated by factors utilizing the activin II B receptor.

The activin type IIB receptor (Acvr2b) is the cell surface receptor for multiple transforming growth factor ß (TGF-ß) superfamily ligands, several of which regulate muscle growth in mammals. To investigate the role of the Acvr2b signaling pathway in the growth and development of skeletal muscle in teleost fish, transgenic rainbow trout (RBT; Oncorhynchus mykiss) expressing a truncated form of the acvr2b-2a (acvr2b(▵)) in muscle tissue were produced. High levels of acvr2b(▵) expression were detected in the majority of P1 transgenic fish. Transgenic P1 trout developed enhanced, localized musculature in both the epaxial and hypaxial regions (dubbed 'six pack'). The F1 transgenic offspring did not exhibit localized muscle growth, but rather developed a uniform body morphology with greater girth, condition factor and increased muscle fiber hypertrophy. There was a high degree of variation in the mass of both P1 and F1 transgenic fish, with several fish of each generation exhibiting enhanced growth compared with other transgenic and control siblings. The 'six pack' phenotype observed in P1 transgenic RBT overexpressing acvr2b(▵) and the presence of F1 individuals with altered muscle morphology provides compelling evidence for the importance of TGF-ß signaling molecules in regulating muscle growth in teleost fish.

Overexpression of follistatin in trout stimulates increased muscling.

Deletion or inhibition of myostatin in mammals has been demonstrated to markedly increase muscle mass by hyperplasia, hypertrophy, or a combination of both. Despite a remarkably high degree of conservation with the mammalian protein, the function of myostatin remains unknown in fish, many species of which continue muscle growth throughout the lifecycle by hyperplasia. Transgenic rainbow trout (Oncorhynchus mykiss) overexpressing follistatin, one of the more efficacious antagonists of myostatin, were produced to investigate the effect of this protein on muscle development and growth. P(1) transgenics overexpressing follistatin in muscle tissue exhibited increased epaxial and hypaxial muscling similar to that observed in double-muscled cattle and myostatin null mice. The hypaxial muscling generated a phenotype reminiscent of well-developed rectus abdominus and intercostal muscles in humans and was dubbed "six pack." Body conformation of the transgenic animals was markedly altered, as measured by condition factor, and total muscle surface area increased. The increased muscling was due almost exclusively to hyperplasia as evidenced by a higher number of fibers per unit area and increases in the percentage of smaller fibers and the number of total fibers. In several individuals, asymmetrical muscling was observed, but no changes in mobility or behavior of follistatin fish were observed. The findings indicate that overexpression of follistatin in trout, a species with indeterminate growth rate, enhances muscle growth. It remains to be determined whether the double muscling in trout is due to inhibition of myostatin, other growth factors, or both.

Oncolytic Virus-Mediated RAS Targeting in Rhabdomyosarcoma.

Aberrant activation of the receptor tyrosine kinase-mediated RAS signaling cascade is the primary driver of embryonal rhabdomyosarcoma (ERMS), a pediatric cancer characterized by a block in myogenic differentiation. To investigate the cellular function of activated RAS signaling in regulating the growth and differentiation of ERMS cells, we genetically ablated activated RAS oncogenes with high-efficiency genome-editing technology. Knockout of NRAS in CRISPR-inducible ERMS xenograft models resulted in near-complete tumor regression through a combination of cell death and myogenic differentiation. Utilizing this strategy for therapeutic RAS targeting in ERMS, we developed a recombinant oncolytic myxoma virus (MYXV) engineered with CRISPR/Cas9 gene-editing capability. Treatment of pre-clinical human ERMS tumor xenografts with an NRAS-targeting version of this MYXV significantly reduced tumor growth and increased overall survival. Our data suggest that targeted gene-editing cancer therapies have promising translational applications, especially with improvements to gene-targeting specificity and oncolytic vector technology.

Corrigendum: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy.

This corrects the article DOI: 10.1038/ncomms14454.

Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy.

Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx4cv mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.