RESUMO
CRISPR-based technologies have rapidly enabled the democratization of genome editing in academic institutions through distribution by Addgene over the past decade. Recently, several distribution milestones have been reached, with a collection of >15,000 plasmids deposited by >1,000 laboratories spanning â¼40 countries now shipped 300,000 times to â¼5,000 organizations traversing â¼100 countries. Yet, both deposits of and requests for CRISPR plasmids continue to rise for this disruptive technology. Distribution patterns revealed robust demand for three distinct classes of CRISPR effectors, namely nucleases (e.g., Cas9 and Cas12), modulators (deactivated CRISPR nucleases fused to transcriptional regulators and epigenome modifiers), and chimeric effectors (Cas proteins fused to enzymes carrying out other activities such as deamination, reverse transcription, transposition, and integration). Yearly deposits over the past decade are requested in near-even proportions, reflecting continuous technological development and requests for novel constructs. Though it is unclear whether the slowing rate of requests is inherent to a pandemic operational lag or a transition from emerging to mature technology, it is noteworthy that the relative proportion of requests from plasmids deposited in the previous year remains stable, suggesting robust development of novel tools concurrent with continued adoption of editing, base editing, prime editing, and more. Predictably, most requested plasmids are designed for mammalian genome manipulation, presumably for medical research and human health pursuits, reflecting investments in therapeutic applications. Concurrently, requests for plant and microbial constructs are on the rise, especially in regions of the world more reliant on local agricultural inputs and focused on food and feed applications, illustrating continued diversification of genome editing applications.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Humanos , Sistemas CRISPR-Cas/genética , Plantas/genética , Genoma , Plasmídeos/genética , Mamíferos/genéticaRESUMO
Mechanisms driving the prolonged meiotic prophase I in mammals are poorly understood. RNA helicase YTHDC2 is critical for mitosis to meiosis transition. However, YTHDC2 is highly expressed in pachytene cells. Here we identify an essential role for YTHDC2 in meiotic progression. Specifically, YTHDC2 deficiency causes microtubule-dependent telomere clustering and apoptosis at the pachytene stage of prophase I. Depletion of YTHDC2 results in a massively dysregulated transcriptome in pachytene cells, with a tendency toward upregulation of genes normally expressed in mitotic germ cells and downregulation of meiotic transcripts. Dysregulation does not correlate with m6A status, and YTHDC2-bound mRNAs are enriched in genes upregulated in mutant germ cells, revealing that YTHDC2 primarily targets mRNAs for degradation. Furthermore, altered transcripts in mutant pachytene cells encode microtubule network proteins. Our results demonstrate that YTHDC2 regulates the pachytene stage by perpetuating a meiotic transcriptome and preventing microtubule network changes that could lead to telomere clustering.
Assuntos
Meiose , Microtúbulos/fisiologia , Estágio Paquíteno , RNA Helicases/fisiologia , Espermatócitos/citologia , Telômero , Transcriptoma , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espermatócitos/metabolismoRESUMO
The N6-methyladenosine (m6A) modification is the most prevalent internal RNA modification in eukaryotes. The majority of m6A sites are found in the last exon and 3' UTRs. Here we show that the nuclear m6A reader YTHDC1 is essential for embryo viability and germline development in mouse. Specifically, YTHDC1 is required for spermatogonial development in males and for oocyte growth and maturation in females; Ythdc1-deficient oocytes are blocked at the primary follicle stage. Strikingly, loss of YTHDC1 leads to extensive alternative polyadenylation in oocytes, altering 3' UTR length. Furthermore, YTHDC1 deficiency causes massive alternative splicing defects in oocytes. The majority of splicing defects in mutant oocytes are rescued by introducing wild-type, but not m6A-binding-deficient, YTHDC1. YTHDC1 is associated with the pre-mRNA 3' end processing factors CPSF6, SRSF3, and SRSF7. Thus, YTHDC1 plays a critical role in processing of pre-mRNA transcripts in the oocyte nucleus and may have similar non-redundant roles throughout fetal development.
Assuntos
Processamento Alternativo/genética , Camundongos/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Oócitos/crescimento & desenvolvimento , Poliadenilação/genética , Fatores de Processamento de RNA/genética , Regiões 3' não Traduzidas/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Núcleo Celular/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Desenvolvimento Embrionário/genética , Éxons/genética , Feminino , Masculino , Camundongos/genética , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/metabolismo , Oócitos/metabolismo , Precursores de RNA/genética , Fatores de Processamento de RNA/deficiência , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Espermatogônias/crescimento & desenvolvimento , Espermatogônias/metabolismoRESUMO
The microrchidia (MORC) family proteins are chromatin-remodelling factors and function in diverse biological processes such as DNA damage response and transposon silencing. Here, we report that mouse Morc2b encodes a functional germ cell-specific member of the MORC protein family. Morc2b arose specifically in the rodent lineage through retrotransposition of Morc2a during evolution. Inactivation of Morc2b leads to meiotic arrest and sterility in both sexes. Morc2b-deficient spermatocytes and oocytes exhibit failures in chromosomal synapsis, blockades in meiotic recombination, and increased apoptosis. Loss of MORC2B causes mis-regulated expression of meiosis-specific genes. Furthermore, we find that MORC2B interacts with MORC2A, its sequence paralogue. Our results demonstrate that Morc2b, a relatively recent gene, has evolved an essential role in meiosis and fertility.
Assuntos
Fertilidade/genética , Meiose/genética , Fatores de Transcrição/fisiologia , Animais , Pareamento Cromossômico/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/metabolismo , Homologia de Sequência , Espermatócitos/metabolismo , Fatores de Transcrição/genéticaRESUMO
The germ cell lineage is specified early in embryogenesis and undergoes complex developmental programs to generate gametes. Here, we conducted genetic studies to investigate the role of Utf1 (Undifferentiated embryonic cell transcription factor 1) in mouse germ cell development. Utf1 is expressed in pluripotent embryonic stem (ES) cells and regulates ES cell differentiation. In a proteomics screen, we identified UTF1 among 38 proteins including DNMT3L and DND1 that associate with chromatin in embryonic testes. We find that UTF1 is expressed in embryonic and newborn gonocytes and in a subset of early spermatogonia. Ubiquitous inactivation of Utf1 causes embryonic lethality in mice with a hybrid genetic background. Male mice with a germline-specific deletion of Utf1 resulting from Prdm1-Cre mediated recombination are born with significantly fewer gonocytes and exhibit defective spermatogenesis and reduced sperm count as young adults. These defects are ameliorated in older animals. These results demonstrate that UTF1 is required for embryonic development and regulates male germ cell development.
Assuntos
Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Espermatozoides/metabolismo , Transativadores/deficiência , Transativadores/genética , Animais , Linhagem da Célula/genética , Cromatina/metabolismo , Deleção de Genes , Masculino , Camundongos , Proteômica , Espermatogênese , Espermatozoides/citologiaRESUMO
BACKGROUND: An enduring question surrounding sex chromosome evolution is whether effective hemizygosity in the heterogametic sex leads inevitably to dosage compensation of sex-linked genes, and whether this compensation has been observed in a variety of organisms. Incongruence in the conclusions reached in some recent reports has been attributed to different high-throughput approaches to transcriptome analysis. However, recent reports each utilizing RNA-seq to gauge X-linked gene expression relative to autosomal gene expression also arrived at diametrically opposed conclusions regarding X chromosome dosage compensation in mammals. RESULTS: Here we analyze RNA-seq data from X-monosomic female human and mouse tissues, which are uncomplicated by genes that escape X-inactivation, as well as published RNA-seq data to describe relative X expression (RXE). We find that the determination of RXE is highly dependent upon a variety of computational, statistical and biological assumptions underlying RNA-seq analysis. Parameters implemented in short-read mapping programs, choice of reference genome annotation, expression data distribution, tissue source for RNA and RNA-seq library construction method have profound effects on comparing expression levels across chromosomes. CONCLUSIONS: Our analysis shows that the high number of paralogous gene families on the mammalian X chromosome relative to autosomes contributes to the ambiguity in RXE calculations, RNA-seq analysis that takes into account that single- and multi-copy genes are compensated differently supports the conclusion that, in many somatic tissues, the mammalian X is up-regulated compared to the autosomes.