Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.

CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.

The arginine methyltransferase Prmt1 coordinates the germline arginine methylome essential for spermatogonial homeostasis and male fertility.

Arginine methylation, catalyzed by the protein arginine methyltransferases (PRMTs), is a common post-translational protein modification (PTM) that is engaged in a plethora of biological events. However, little is known about how the methylarginine-directed signaling functions in germline development. In this study, we discover that Prmt1 is predominantly distributed in the nuclei of spermatogonia but weakly in the spermatocytes throughout mouse spermatogenesis. By exploiting a combination of three Cre-mediated Prmt1 knockout mouse lines, we unravel that Prmt1 is essential for spermatogonial establishment and maintenance, and that Prmt1-catalyzed asymmetric methylarginine coordinates inherent transcriptional homeostasis within spermatogonial cells. In conjunction with high-throughput CUT&Tag profiling and modified mini-bulk Smart-seq2 analyses, we unveil that the Prmt1-deposited H4R3me2a mark is permissively enriched at promoter and exon/intron regions, and sculpts a distinctive transcriptomic landscape as well as the alternative splicing pattern, in the mouse spermatogonia. Collectively, our study provides the genetic and mechanistic evidence that connects the Prmt1-deposited methylarginine signaling to the establishment and maintenance of a high-fidelity transcriptomic identity in orchestrating spermatogonial development in the mammalian germline.

Generalizable transcriptome-based tumor malignant level evaluation and molecular subtyping towards precision oncology.

In cancer treatment, therapeutic strategies that integrate tumor-specific characteristics (i.e., precision oncology) are widely implemented to provide clinical benefits for cancer patients. Here, through in-depth integration of tumor transcriptome and patients' prognoses across cancers, we investigated dysregulated and prognosis-associated genes and catalogued such important genes in a cancer type-dependent manner. Utilizing the expression matrices of these genes, we built models to quantitatively evaluate the malignant levels of tumors across cancers, which could add value to the clinical staging system for improved prediction of patients' survival. Furthermore, we performed a transcriptome-based molecular subtyping on hepatocellular carcinoma, which revealed three subtypes with significantly diversified clinical outcomes, mutation landscapes, immune microenvironment, and dysregulated pathways. As tumor transcriptome was commonly profiled in clinical practice with low experimental complexity and cost, this work proposed easy-to-perform approaches for practical clinical promotion towards better healthcare and precision oncology of cancer patients.

Building RNA-protein germ granules: insights from the multifaceted functions of DEAD-box helicase Vasa/Ddx4 in germline development.

The segregation and maintenance of a dedicated germline in multicellular organisms is essential for species propagation in the sexually reproducing metazoan kingdom. The germline is distinct from somatic cells in that it is ultimately dedicated to acquiring the "totipotency" and to regenerating the offspring after fertilization. The most striking feature of germ cells lies in the presence of characteristic membraneless germ granules that have recently proven to behave like liquid droplets resulting from liquid-liquid phase separation (LLPS). Vasa/Ddx4, a faithful DEAD-box family germline marker highly conserved across metazoan species, harbors canonical DEAD-box motifs and typical intrinsically disordered sequences at both the N-terminus and C-terminus. This feature enables it to serve as a primary driving force behind germ granule formation and helicase-mediated RNA metabolism (e.g., piRNA biogenesis). Genetic ablation of Vasa/Ddx4 or the catalytic-dead mutations abolishing its helicase activity led to sexually dimorphic germline defects resulting in either male or female sterility among diverse species. While recent efforts have discovered pivotal functions of Vasa/Ddx4 in somatic cells, especially in multipotent stem cells, we herein summarize the helicase-dependent and -independent functions of Vasa/Ddx4 in the germline, and discuss recent findings of Vasa/Ddx4-mediated phase separation, germ granule formation and piRNA-dependent retrotransposon control essential for germline development.

The Spin1 interactor, Spindoc, is dispensable for meiotic division, but essential for haploid spermatid development in mice.

In mammals, germline development undergoes dramatic morphological and molecular changes and is epigenetically subject to intricate yet exquisite regulation. Which epigenetic players and how they participate in the germline developmental process are not fully characterized. Spin1 is a multifunctional epigenetic protein reader that has been shown to recognize H3 "K4me3-R8me2a" histone marks, and more recently the non-canonical bivalent H3 "K4me3-K9me3/2" marks as well. As a robust Spin1-interacting cofactor, Spindoc has been identified to enhance the binding of Spin1 to its substrate histone marks, thereby modulating the downstream signaling; However, the physiological role of Spindoc in germline development is unknown. We generated two Spindoc knockout mouse models through CRISPR/Cas9 strategy, which revealed that Spindoc is specifically required for haploid spermatid development, but not essential for meiotic divisions in spermatocytes. This study unveiled a new epigenetic player that participates in haploid germline development.

Npat-dependent programmed Sertoli cell proliferation is indispensable for testis cord development and germ cell mitotic arrest.

As the major somatic cell type, Sertoli cells undergo active proliferation and play essential roles to establish testis cord at fetal stage. They also function to maintain germ cell development throughout the life of testicular development. However, the significance of Sertoli cell number for testis cord development and gonocyte fate is still unclear. Nuclear protein ataxia-telangiectasia (NPAT, also known as p220), a substrate of cyclin E/cyclin-dependent kinase 2, is well known as a regulator of cell proliferation through regulating histone expression. To study the role of NPAT during Sertoli cell development, we generated a mouse strain carrying conditional floxed Npat alleles, when crossing with anti-Müllerian hormone-cre, leading to the specific deletion of Npat in Sertoli cells. Npat disruption in Sertoli cells inhibited the programmed proliferation of fetal Sertoli cells resulting in disruption of developing testis cords, and subsequent postnatal mutant testes were severely hypoplastic. Germ cells, which are presumed to be in quiescent status during perinatal stage, exited G0 phase arrest and re-enter mitotic cell cycle prematurely. Of particular note, some germ cells possessed the meiotic signal in Npat-deficient testes. Our data thus indicates that the function of Npat-dependent Sertoli cells is essential at multiple steps in testis development, and this study also identifies Sertoli cells as a major regulator of germ cell development, which are required to maintain a local growth niche to repress premature mitosis and meiosis of gonocytes.-Jiang, X., Yin, S., Fan, S., Bao, J., Jiao, Y., Ali, A., Iqbal, F., Xu, J., Zhang, Y., Shi, Q. Npat-dependent programmed Sertoli cell proliferation is indispensable for testis cord development and germ cell mitotic arrest.

The arginine methyltransferase CARM1 represses p300•ACT•CREMτ activity and is required for spermiogenesis.

CARM1 is a protein arginine methyltransferase (PRMT) that has been firmly implicated in transcriptional regulation. However, the molecular mechanisms by which CARM1 orchestrates transcriptional regulation are not fully understood, especially in a tissue-specific context. We found that Carm1 is highly expressed in the mouse testis and localizes to the nucleus in spermatids, suggesting an important role for Carm1 in spermiogenesis. Using a germline-specific conditional Carm1 knockout mouse model, we found that it is essential for the late stages of haploid germ cell development. Loss of Carm1 led to a low sperm count and deformed sperm heads that can be attributed to defective elongation of round spermatids. RNA-seq analysis of Carm1-null spermatids revealed that the deregulated genes fell into similar categories as those impacted by p300-loss, thus providing a link between Carm1 and p300. Importantly, p300 has long been known to be a major Carm1 substrate. We found that CREMτ, a key testis-specific transcription factor, associates with p300 through its activator, ACT, and that this interaction is negatively regulated by the methylation of p300 by Carm1. Thus, high nuclear Carm1 levels negatively impact the p300•ACT•CREMτ axis during late stages of spermiogenesis.

Identification of Rpl29 as a major substrate of the lysine methyltransferase Set7/9.

Set7/9 (also known as Set7, Set9, Setd7, and Kmt7) is a lysine methyltransferase that catalyzes the methylation of multiple substrates, including histone H3 and non-histone proteins. Although not essential for normal development and physiology, Set7/9-mediated methylation events play important roles in regulating cellular pathways involved in various human diseases, making Set7/9 a promising therapeutic target. Multiple Set7/9 inhibitors have been developed, which exhibit varying degrees of potency and selectivity in vitro However, validation of these compounds in vivo has been hampered by the lack of a reliable cellular biomarker for Set7/9 activity. Here, we report the identification of Rpl29, a ribosomal protein abundantly expressed in all cell types, as a major substrate of Set7/9. We show that Rpl29 lysine 5 (Rpl29K5) is methylated exclusively by Set7/9 and can be demethylated by Lsd1 (also known as Kdm1a). Rpl29 is not a core component of the ribosome translational machinery and plays a regulatory role in translation efficiency. Our results indicate that Rpl29 methylation has no effect on global protein synthesis but affects Rpl29 subcellular localization. Using an Rpl29 methylation-specific antibody, we demonstrate that Rpl29K5 methylation is present ubiquitously and validate that (R)-PFI-2, a Set7/9 inhibitor, efficiently reduces Rpl29K5 methylation in cell lines. Thus, Rpl29 methylation can serve as a specific cellular biomarker for measuring Set7/9 activity.

Sperm-borne miRNAs and endo-siRNAs are important for fertilization and preimplantation embryonic development.

Although it is believed that mammalian sperm carry small noncoding RNAs (sncRNAs) into oocytes during fertilization, it remains unknown whether these sperm-borne sncRNAs truly have any function during fertilization and preimplantation embryonic development. Germline-specific Dicer and Drosha conditional knockout (cKO) mice produce gametes (i.e. sperm and oocytes) partially deficient in miRNAs and/or endo-siRNAs, thus providing a unique opportunity for testing whether normal sperm (paternal) or oocyte (maternal) miRNA and endo-siRNA contents are required for fertilization and preimplantation development. Using the outcome of intracytoplasmic sperm injection (ICSI) as a readout, we found that sperm with altered miRNA and endo-siRNA profiles could fertilize wild-type (WT) eggs, but embryos derived from these partially sncRNA-deficient sperm displayed a significant reduction in developmental potential, which could be rescued by injecting WT sperm-derived total or small RNAs into ICSI embryos. Disrupted maternal transcript turnover and failure in early zygotic gene activation appeared to associate with the aberrant miRNA profiles in Dicer and Drosha cKO spermatozoa. Overall, our data support a crucial function of paternal miRNAs and/or endo-siRNAs in the control of the transcriptomic homeostasis in fertilized eggs, zygotes and two-cell embryos. Given that supplementation of sperm RNAs enhances both the developmental potential of preimplantation embryos and the live birth rate, it might represent a novel means to improve the success rate of assisted reproductive technologies in fertility clinics.

UPF2-Dependent Nonsense-Mediated mRNA Decay Pathway Is Essential for Spermatogenesis by Selectively Eliminating Longer 3'UTR Transcripts.

During transcription, most eukaryotic genes generate multiple alternative cleavage and polyadenylation (APA) sites, leading to the production of transcript isoforms with variable lengths in the 3' untranslated region (3'UTR). In contrast to somatic cells, male germ cells, especially pachytene spermatocytes and round spermatids, express a distinct reservoir of mRNAs with shorter 3'UTRs that are essential for spermatogenesis and male fertility. However, the mechanisms underlying the enrichment of shorter 3'UTR transcripts in the developing male germ cells remain unknown. Here, we report that UPF2-mediated nonsense-mediated mRNA decay (NMD) plays an essential role in male germ cells by eliminating ubiquitous genes-derived, longer 3'UTR transcripts, and that this role is independent of its canonical role in degrading "premature termination codon" (PTC)-containing transcripts in somatic cell lineages. This report provides physiological evidence supporting a noncanonical role of the NMD pathway in achieving global 3'UTR shortening in the male germ cells during spermatogenesis.

UPF2, a nonsense-mediated mRNA decay factor, is required for prepubertal Sertoli cell development and male fertility by ensuring fidelity of the transcriptome.

Nonsense-mediated mRNA decay (NMD) represents a highly conserved RNA surveillance mechanism through which mRNA transcripts bearing premature termination codons (PTCs) are selectively degraded to maintain transcriptomic fidelity in the cell. Numerous in vitro studies have demonstrated the importance of the NMD pathway; however, evidence supporting its physiological necessity has only just started to emerge. Here, we report that ablation of Upf2, which encodes a core NMD factor, in murine embryonic Sertoli cells (SCs) leads to severe testicular atrophy and male sterility owing to rapid depletion of both SCs and germ cells during prepubertal testicular development. RNA-Seq and bioinformatic analyses revealed impaired transcriptomic homeostasis in SC-specific Upf2 knockout testes, characterized by an accumulation of PTC-containing transcripts and the transcriptome-wide dysregulation of genes encoding splicing factors and key proteins essential for SC fate control. Our data demonstrate an essential role of UPF2-mediated NMD in prepubertal SC development and male fertility.

Spata6 is required for normal assembly of the sperm connecting piece and tight head-tail conjunction.

"Pinhead sperm," or "acephalic sperm," a type of human teratozoospermia, refers to the condition in which ejaculate contains mostly sperm flagella without heads. Family clustering and homogeneity of this syndrome suggests a genetic basis, but the causative genes remain largely unknown. Here we report that Spata6, an evolutionarily conserved testis-specific gene, encodes a protein required for formation of the segmented columns and the capitulum, two major structures of the sperm connecting piece essential for linking the developing flagellum to the head during late spermiogenesis. Inactivation of Spata6 in mice leads to acephalic spermatozoa and male sterility. Our proteomic analyses reveal that SPATA6 is involved in myosin-based microfilament transport through interaction with myosin subunits (e.g., MYL6).

RAN-binding protein 9 is involved in alternative splicing and is critical for male germ cell development and male fertility.

As a member of the large Ran-binding protein family, Ran-binding protein 9 (RANBP9) has been suggested to play a critical role in diverse cellular functions in somatic cell lineages in vitro, and this is further supported by the neonatal lethality phenotype in Ranbp9 global knockout mice. However, the exact molecular actions of RANBP9 remain largely unknown. By inactivation of Ranbp9 specifically in testicular somatic and spermatogenic cells, we discovered that Ranbp9 was dispensable for Sertoli cell development and functions, but critical for male germ cell development and male fertility. RIP-Seq and proteomic analyses revealed that RANBP9 was associated with multiple key splicing factors and directly targeted >2,300 mRNAs in spermatocytes and round spermatids. Many of the RANBP9 target and non-target mRNAs either displayed aberrant splicing patterns or were dysregulated in the absence of Ranbp9. Our data uncovered a novel role of Ranbp9 in regulating alternative splicing in spermatogenic cells, which is critical for normal spermatogenesis and male fertility.

Two miRNA clusters, miR-34b/c and miR-449, are essential for normal brain development, motile ciliogenesis, and spermatogenesis.

Ablation of a single miRNA gene rarely leads to a discernable developmental phenotype in mice, in some cases because of compensatory effects by other functionally related miRNAs. Here, we report that simultaneous inactivation of two functionally related miRNA clusters (miR-34b/c and miR-449) encoding five miRNAs (miR-34b, miR-34c, miR-449a, miR-449b, and miR-449c) led to sexually dimorphic, partial perinatal lethality, growth retardation, and infertility. These developmental defects correlated with the dysregulation of ∼ 240 target genes, which are mainly involved in three major cellular functions, including cell-fate control, brain development and microtubule dynamics. Our data demonstrate an essential role of a miRNA family in brain development, motile ciliogenesis, and spermatogenesis.

Epigenetic regulation of the histone-to-protamine transition during spermiogenesis.

In mammals, male germ cells differentiate from haploid round spermatids to flagella-containing motile sperm in a process called spermiogenesis. This process is distinct from somatic cell differentiation in that the majority of the core histones are replaced sequentially, first by transition proteins and then by protamines, facilitating chromatin hyper-compaction. This histone-to-protamine transition process represents an excellent model for the investigation of how epigenetic regulators interact with each other to remodel chromatin architecture. Although early work in the field highlighted the critical roles of testis-specific transcription factors in controlling the haploid-specific developmental program, recent studies underscore the essential functions of epigenetic players involved in the dramatic genome remodeling that takes place during wholesale histone replacement. In this review, we discuss recent advances in our understanding of how epigenetic players, such as histone variants and histone writers/readers/erasers, rewire the haploid spermatid genome to facilitate histone substitution by protamines in mammals.

A novel class of somatic small RNAs similar to germ cell pachytene PIWI-interacting small RNAs.

PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a yet to be identified pathway, which is different from all currently known small RNA biogenetic pathways. In addition, pilRNAs appear to preferentially target the 3'-UTRs of mRNAs in a partially complementary manner. Our data suggest that pilRNAs, as an integral component of the small RNA transcriptome in somatic cell lineages, represent a distinct population of small RNAs that may have functions similar to germ cell piRNAs.

Untacking small RNA profiling and RNA fragment footprinting: Approaches and challenges in library construction.

Small RNAs (sRNAs) with sizes ranging from 15 to 50 nucleotides (nt) are critical regulators of gene expression control. Prior studies have shown that sRNAs are involved in a broad range of biological processes, such as organ development, tumorigenesis, and epigenomic regulation; however, emerging evidence unveils a hidden layer of diversity and complexity of endogenously encoded sRNAs profile in eukaryotic organisms, including novel types of sRNAs and the previously unknown post-transcriptional RNA modifications. This underscores the importance for accurate, unbiased detection of sRNAs in various cellular contexts. A multitude of high-throughput methods based on next-generation sequencing (NGS) are developed to decipher the sRNA expression and their modifications. Nonetheless, distinct from mRNA sequencing, the data from sRNA sequencing suffer frequent inconsistencies and high variations emanating from the adapter contaminations and RNA modifications, which overall skew the sRNA libraries. Here, we summarize the sRNA-sequencing approaches, and discuss the considerations and challenges for the strategies and methods of sRNA library construction. The pros and cons of sRNA sequencing have significant implications for implementing RNA fragment footprinting approaches, including CLIP-seq and Ribo-seq. We envision that this review can inspire novel improvements in small RNA sequencing and RNA fragment footprinting in future. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs.

Immunological regulation by Toll-1 and Spätzle-4 in larval density-dependent prophylaxis of the oriental armyworm, Mythimna separata.

High population density has been shown to alter insect prophylactic immunity. Toll-Spätzle pathway performs a key function in insect innate immune response. To determine the role of Toll and Spätzle, two main components of Toll-Spätzle pathway, in the density-dependent prophylaxis of Mythimna separata. We identified full-length cDNA encoding the Toll-1 and Spätzle-4 genes in M. separata (designed MsToll-1 and Ms Spätzle-4). Both MsToll-1 and MsSpätzle-4 were expressed throughout all developmental stages. MsToll-1 expression was highly in fat body and brain and MsSpätzle-4 was highly expressed in brain and Malpighian tubule. With increased larval density, MsToll-1 expression was markedly up-regulated. MsSpätzle-4 expression was found to be raised in larvae that were fed in high density (5 and 10 larvae per jar). Co-immunoprecipitation assays demonstrated that MsToll-1 interacted with MsSpätzle-4. Immune-related genes transcriptions were considerably reduced in high-density larvae MsToll-1 (or MsSpätzle-4) was silenced by dsRNA injection. Meanwhile, a discernible reduction in the survival rate of the larvae exposed to Bacillus thuringiensis infection with silence of MsToll-1 (or MsSpätzle-4) was observed. This study implies that prophylactic immunity was influenced by crowded larvae via modulating the Toll-Spätzle pathway in M. separata and allow for a new understanding of into density-dependent prophylaxis in insects.

Incomplete cre-mediated excision leads to phenotypic differences between Stra8-iCre; Mov10l1(lox/lox) and Stra8-iCre; Mov10l1(lox/Δ) mice.

In the Cre-loxp system, expression level and activity of Cre recombinase in a Cre deleter line are critical because these determine not only the cell specificity of gene knockout (KO), but also the efficiency of Cre-mediated excision in a specific cell lineage. Although the spatiotemporal expression pattern of a Cre transgene is usually defined upon the generation of the mouse line, the Cre excision efficiency in a specific targeted cell lineage is rarely evaluated and often assumed to be 100%. Incomplete excision can lead to highly variable phenotypes owing to mosaicism (i.e., coexistence of cells with the flox or the recombined flox allele) and this problem has long been overlooked. Here, we report that Stra8-codon-improved Cre recombinase (iCre), a transgenic allele expressing iCre under the control of the male germ cell-specific Stra8 promoter, could efficiently delete one Mov10l1 flox allele in spermatogenic cells, whereas the excision was incomplete when two Mov10l1 flox alleles were present. The incomplete Cre-mediated excision led to a testicular phenotype that was much less severe than that in the true conditional KO (inactivation, 100%) mice. Our findings suggest that it is essential to determine the efficiency of Cre excision when Cre-loxp system is used for deleting genes in a specific cell lineage and the Cre; gene(lox) (/)(Δ) genotype should be used to evaluate phenotypes instead of Cre; gene(lox/lox) owing to the fact that the latter usually bears incomplete deletion of the flox allele(s).

MicroRNA-449 and microRNA-34b/c function redundantly in murine testes by targeting E2F transcription factor-retinoblastoma protein (E2F-pRb) pathway.

MicroRNAs (miRNAs) mainly function as post-transcriptional regulators and are involved in a wide range of physiological and pathophysiological processes such as cell proliferation, differentiation, apoptosis, and tumorigenesis. Mouse testes express a large number of miRNAs. However, the physiological roles of these testicular miRNAs remain largely unknown. Using microarray and quantitative real time PCR assays, we identified that miRNAs of the microRNA-449 (miR-449) cluster were preferentially expressed in the mouse testis, and their levels were drastically up-regulated upon meiotic initiation during testicular development and in adult spermatogenesis. The expression pattern of the miR-449 cluster resembled that of microRNA-34b/c (miR-34b/c) during spermatogenesis. Further analyses identified that cAMP-responsive element modulator τ and SOX5, two transcription factors essential for regulating male germ cell gene expression, acted as the upstream transactivators to stimulate the expression of the miR-449 cluster in mouse testes. Despite its abundant expression in testicular germ cells, miR-449-null male mice developed normally and exhibited normal spermatogenesis and fertility. Our data further demonstrated that miR-449 shared a cohort of target genes that belong to the E2F transcription factor-retinoblastoma protein pathway with the miR-34 family, and levels of miR-34b/c were significantly up-regulated in miR-449-null testes. Taken together, our data suggest that the miR-449 cluster and miR-34b/c function redundantly in the regulation of male germ cell development in murine testes.