Communication about genetic editing : CRISPR, between optimism and false expectations

Communication is essential in all areas of society, but communication in science is inescapable. Communicating means sharing, showing, teaching, and transferring knowledge about discoveries, observations, and findings both to colleagues and to society in general. That is why good communication must always accompany good science. CRISPR genetic editing tools allow us to modify, at will, the genome of any living organism, including our own species. In this text I review the different relevant communicative events in the short but intense life of these «molecular scissors», so called for their ability to cut the DNA molecule effectively and with precision

La modificación genética dirigida en ratones es premiada con el Nobel de Fisiología o Medicina en 2007

The Nobel Prize in Physiology or Medicine for 2007 has been awarded jointly, well deserved, to the American scientists Mario Capecchi and Oliver Smithies, and to the British scientist Sir Martin Evans, for their pioneer contributions and experimental design resulting in the obtention of the first knockout mice with a gene targeted event, with the specific inactivation of a gene, leaving the rest of the genome intact. Martin Evans described, in 1981, the extraordinary plasticity of pluripotent embryonic stem cells, from the inner cell mass of the blastocyst, thus allowing their maintenance in culture indefinitely, their genetic manipulation and, eventually, their reintroduction in a new blastocyst, without loosing their capacity to differentiate to any of the cellular types found in an adult organism, including the germ line. Mario Capecchi explored, in the 80s, the strategies that allowed him to selectively alter a given genetic sequence, using the homologous recombination procedure, and established, in 1988, the general method of positive-negative double selection. Finally, Oliver Smithies, in 1989, modified a first gene (inactivated) in embryonic stem cells in culture, later obtained a chimera and eventually, through a number of crosses, an animal that, in all of its cells, was carrying the initial mutation of the selected gene.Key Words: embryonic stem cells, knockout mice, blastocysts, homologous recombination, functional genomics.El Premio Nobel de Fisiología o Medicina 2007 ha sido otorgado, merecidamente, a los investigadores norteamericanos Mario Capecchi y Oliver Smithies, y al científico británico Sir Martin Evans, por sus contribuciones pioneras y diseño experimental conducentes a la obtención de los primeros ratones mutantes con una modificación genética dirigida, con la inactivación específica de un gen, dejando intacto el resto del genoma. Martin Evans describió, en 1981, la extraordinaria plasticidad de las células troncales embrionales pluripotentes de la masa interna celular del blastocisto, lo que permitía mantenerlas en cultivo indefinidamente, modificarlas genéticamente y reintroducirlas en un nuevo blastocisto, sin que perdieran la posibilidad de convertirse en cualquiera de los tipos celulares que pueblan un organismo adulto, incluyendo la línea germinal. Mario Capecchi exploró, en los años 80, las estrategias que permitieron modificar, de forma selectiva, una determinada secuencia genética, mediante el procedimiento de recombinación homóloga y estableció en 1988 el método general de selección doble positiva-negativa. Finalmente, Oliver Smithies, en 1989, fue el primer investigador que integró las evidencias experimentales de sus dos colegas, modificó un gen (lo inactivó) en células troncales embrionales pluripotentes en cultivo, obtuvo después un ratón quimérico y finalmente, mediante cruces, un animal que, en todas sus células, era portador de la mutación inicial del gen seleccionado.Palabras clave: células troncales embrionarias, ratones mutantes, blastocistos, recombinación homóloga, genómica funcional

The INFRAFRONTIER Research Infrastructure

INFRAFRONTIER proporciona recursos científicos únicos, servicios y experiencia para avanzar en la comprensión y el tratamiento de enfermedades humanas utilizando el modelo roedor. Fomenta la investigación responsable y reproducible, minimizando la duplicidad proporcionando recursos centralizados de alta calidad. INFRAFRONTIER es una infraestructura para generación de fenotipado, archivo y distribución de modelos mamíferos.N

Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR-Cas9-mediated mutagenesis

Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system, allow simple and rapid genetic modification in most model organisms and human cell lines. Here, we report the production and analysis of mice carrying the inactivation via deletion of a genomic insulator, a key non-coding regulatory DNA element found 5′ upstream of the mouse tyrosinase (Tyr) gene. Targeting sequences flanking this boundary in mouse fertilized eggs resulted in the efficient deletion or inversion of large intervening DNA fragments delineated by the RNA guides. The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus. Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact. This study reports how non-coding DNA elements, even if located in repeat-rich genomic sequences, can be efficiently and functionally evaluated in vivo and, furthermore, it illustrates how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be systematically validate

The triennial International Pigment Cell Conference (IPCC)

The International Federation of Pigment Cell Societies (IFPCS) held its XXIII triennial International Pigment Cell Conference (IPCC) in Denver, Colorado in August 2017. The goal of the summit was to provide a venue promoting a vibrant interchange among leading basic and clinical researchers working on leading-edge aspects of melanocyte biology and disease. The philosophy of the meeting, entitled Breakthroughs in Pigment Cell and Melanoma Research, was to deliver a comprehensive program in an inclusive environment fostering scientific exchange and building new academic bridges. This document provides an outlook on the history, accomplishments, and sustainability of the pigment cell and melanoma research community. Shared progress in the understanding of cellular homeostasis of pigment cells but also clinical successes and hurdles in the treatment of melanoma and dermatological disorders continue to drive future research activities. A sustainable direction of the societies creates an international forum identifying key areas of imminent needs in laboratory research and clinical care and ensures the future of this vibrant, diverse and unique research community at the same time. Important advances showcase wealth and breadth of the field in melanocyte and melanoma research and include emerging frontiers in melanoma immunotherapy, medical and surgical oncology, dermatology, vitiligo, albinism, genomics and systems biology, precision bench-to-bedside approaches, epidemiology, pigment biophysics and chemistry, and evolution. This report recapitulates highlights of the federate meeting agenda designed to advance clinical and basic research frontiers from melanoma and dermatological sciences followed by a historical perspective of the associated societies and conferences

A Slc38a8 Mouse Model of FHONDA Syndrome Faithfully Recapitulates the Visual Deficits of Albinism Without Pigmentation Defects

Purpose: We aimed to generate and phenotype a mouse model of foveal hypoplasia, optic nerve decussation defects, and anterior segment dysgenesis (FHONDA), a rare disease associated with mutations in Slc38a8 that causes severe visual alterations similar to albinism without affecting pigmentation. Methods: The FHONDA mouse model was generated with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology using an RNA guide targeting the Scl38a8 murine locus. The resulting mice were backcrossed to C57BL/6J. Melanin content was measured using spectrophotometry. Retinal cell architecture was analyzed through light and electron microscopy. Retinal projections to the brain were evaluated with anterograde labelling in embryos and adults. Visual function was assessed by electroretinography (ERG) and the optomotor test (OT). Results: From numerous Slc38a8 mouse mutant alleles generated, we selected one that encodes a truncated protein (p.196Pro*, equivalent to p.199Pro* in the human protein) closely resembling a mutant allele described in patients (p.200Gln*). Slc38a8 mutant mice exhibit wild-type eye and coat pigmentation with comparable melanin content. Subcellular abnormalities were observed in retinal pigment epithelium cells of Slc38a8 mutant mice. Anterograde labeling experiments of retinal projections in embryos and adults showed a reduction of ipsilateral fibers. Functional visual analyses revealed a decreased ERG response in scotopic conditions and a reduction of visual acuity in mutant mice measured by OT. Conclusions: Slc38a8 mutant mice recapitulate the phenotype of patients with FHONDA concerning their normal pigmentation and their abnormal visual system, in the latter being a hallmark of all types of albinism. These mice will be helpful in better understanding the pathophysiology of this genetic condition.Funded by the Spanish Ministry of Economy and Competitiveness under BIO2015-70978-R, the Spanish Ministry of Science and Innovation under RTI2018-101223-B-I00, CIBERER and Fundación Ramón Areces to L.M. Additionally, Spanish Ministry of Science and Innovation (FEDER-PID2019-106230RB-I00, 2019) and Generalitat Valenciana IDIFEDER/2017/064, 2017, PROMETEO/2021/024, 2021 supported the work of N.C. Funds from INSERM, Sorbonne Université, Retina France and Genespoir supported the work of A.R., as well as LabEx LIFESENSES (ANR-10-LABX-65) and IHU FOReSIGHT (ANR-18-IAHU-01) for the Institut de la Vision, a doctoral fellowship from the French Ministry of Education and Research to V.C

The PEG13-DMR and brain-specific enhancers dictate imprinted expression within the 8q24 intellectual disability risk locus

Background: Genomic imprinting is the epigenetic marking of genes that results in parent-of-origin monoallelic expression. Most imprinted domains are associated with differentially DNA methylated regions (DMRs) that originate in the gametes, and are maintained in somatic tissues after fertilization. This allelic methylation profile is associated with a plethora of histone tail modifications that orchestrates higher order chromatin interactions. The mouse chromosome 15 imprinted cluster contains multiple brain-specific maternally expressed transcripts including Ago2, Chrac1, Trappc9 and Kcnk9 and a paternally expressed gene, Peg13. The promoter of Peg13 is methylated on the maternal allele and is the sole DMR within the locus. To determine the extent of imprinting within the human orthologous region on chromosome 8q24, a region associated with autosomal recessive intellectual disability, Birk-Barel mental retardation and dysmorphism syndrome, we have undertaken a systematic analysis of allelic expression and DNA methylation of genes mapping within an approximately 2 Mb region around TRAPPC9. Results: Utilizing allele-specific RT-PCR, bisulphite sequencing, chromatin immunoprecipitation and chromosome conformation capture (3C) we show the reciprocal expression of the novel, paternally expressed, PEG13 non-coding RNA and maternally expressed KCNK9 genes in brain, and the biallelic expression of flanking transcripts in a range of tissues. We identify a tandem-repeat region overlapping the PEG13 transcript that is methylated on the maternal allele, which binds CTCF-cohesin in chromatin immunoprecipitation experiments and possesses enhancer-blocker activity. Using 3C, we identify mutually exclusive approximately 58 and 500 kb chromatin loops in adult frontal cortex between a novel brain-specific enhancer, marked by H3K4me1 and H3K27ac, with the KCNK9 and PEG13 promoters which we propose regulates brain-specific expression. Conclusions: We have characterised the molecular mechanism responsible for reciprocal allelic expression of the PEG13 and KCNK9 transcripts. Therefore, our observations may have important implications for identifying the cause of intellectual disabilities associated with the 8q24 locu

Frontiers in Pigment Cell and Melanoma Research

We identify emerging frontiers in clinical and basic research of melanocyte biology and its associated biomedical disciplines. We describe challenges and opportunities in clinical and basic research of normal and diseased melanocytes that impact current approaches to research in melanoma and the dermatological sciences. We focus on four themes: (1) clinical melanoma research, (2) basic melanoma research, (3) clinical dermatology, and (4) basic pigment cell research, with the goal of outlining current highlights, challenges, and frontiers associated with pigmentation and melanocyte biology. Significantly, this document encapsulates important advances in melanocyte and melanoma research including emerging frontiers in melanoma immunotherapy, medical and surgical oncology, dermatology, vitiligo, albinism, genomics and systems biology, epidemiology, pigment biophysics and chemistry, and evolution

Impairment of photoreceptor ribbon synapses in a novel Pomt1 conditional knockout mouse model of dystroglycanopathy

Hypoglycosylation of α-dystroglycan (α-DG) resulting from deficiency of protein O-mannosyltransferase 1 (POMT1) may cause severe neuromuscular dystrophies with brain and eye anomalies, named dystroglycanopathies. The retinal involvement of these disorders motivated us to generate a conditional knockout (cKO) mouse experiencing a Pomt1 intragenic deletion (exons 3–4) during the development of photoreceptors, mediated by the Cre recombinase expressed from the cone-rod homeobox (Crx) gene promoter. In this mouse, retinal α-DG was unglycosylated and incapable of binding laminin. Retinal POMT1 deficiency caused significant impairments in both electroretinographic recordings and optokinetic reflex in Pomt1 cKO mice, and immunohistochemical analyses revealed the absence of β-DG and of the α-DG-interacting protein, pikachurin, in the outer plexiform layer (OPL). At the ultrastructural level, noticeable alterations were observed in the ribbon synapses established between photoreceptors and bipolar cells. Therefore, O-mannosylation of α-DG in the retina carried out by POMT1 is crucial for the establishment of proper synapses at the OPL and transmission of visual information from cones and rods to their postsynaptic neurons.This work was funded by the Institute of Health Carlos III (grants PI12/0157 and PI15/073 to J.C. and J.M.-N., PI13/02098 to P.dlV., and RETICS RD12/0034/0006 to P.dlV.), and by the Comunidad de Madrid (‘VISIONANIMAL’ Biomedicine project S2010/BMD2439 to J.C., P.dlV. and L.M.), all of them cofinanced by the European Regional Development Fund (ERDF/FEDER)

diverse human vh antibody fragments with bio therapeutic properties from the crescendo mouse

Abstract We describe the 'Crescendo Mouse', a human VH transgenic platform combining an engineered heavy chain locus with diverse human heavy chain V, D and J genes, a modified mouse Cγ1 gene and complete 3' regulatory region, in a triple knock-out (TKO) mouse background devoid of endogenous immunoglobulin expression. The addition of the engineered heavy chain locus to the TKO mouse restored B cell development, giving rise to functional B cells that responded to immunization with a diverse response that comprised entirely 'heavy chain only' antibodies. Heavy chain variable (VH) domain libraries were rapidly mined using phage display technology, yielding diverse high-affinity human VH that had undergone somatic hypermutation, lacked aggregation and showed enhanced expression in E. coli. The Crescendo Mouse produces human VH fragments, or Humabody® VH, with excellent bio-therapeutic potential, as exemplified here by the generation of antagonistic Humabody® VH specific for human IL17A and IL17RA