RNA-guided DNA endonucleases derived from CRISPR-Cas adaptive immune systems are widely used as powerful genome-engineering tools. Among the diverse CRISPR-Cas nucleases, the type V-F Cas12f (also ...known as Cas14) proteins are exceptionally compact and associate with a guide RNA to cleave single- and double-stranded DNA targets. Here, we report the cryo-electron microscopy structure of Cas12f1 (also known as Cas14a) in complex with a guide RNA and its target DNA. Unexpectedly, the structure revealed that two Cas12f1 molecules assemble with the single guide RNA to recognize the double-stranded DNA target. Each Cas12f1 protomer adopts a different conformation and plays distinct roles in nucleic acid recognition and DNA cleavage, thereby explaining how the miniature Cas12f1 enzyme achieves RNA-guided DNA cleavage as an “asymmetric homodimer.” Our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and provide a framework for the development of compact genome-engineering tools critical for therapeutic genome editing.
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•Cryo-EM structure of Cas12f in complex with a guide RNA and its target DNA•Type V-F effector complex consisting of a Cas12f dimer and a single guide RNA•Molecular mechanism of the RNA-guided DNA cleavage by the miniature Cas12f•Evolutionary insights into a possible origin of the type V CRISPR-Cas enzymes
Takeda et al. report the cryo-EM structure of Cas12f in complex with a guide RNA and its target DNA. The structure reveals that two Cas12f molecules bind the guide RNA to form an effector complex and provides mechanistic insights into the RNA-guided DNA cleavage by the miniature Cas12f enzyme.
In cycling human endometrium, menstruation is followed by rapid estrogen-dependent growth. Upon ovulation, progesterone and rising cellular cAMP levels activate the transcription factor Forkhead box ...O1 (FOXO1) in endometrial stromal cells (EnSCs), leading to cell cycle exit and differentiation into decidual cells that control embryo implantation. Here we show that FOXO1 also causes acute senescence of a subpopulation of decidualizing EnSCs in an IL-8 dependent manner. Selective depletion or enrichment of this subpopulation revealed that decidual senescence drives the transient inflammatory response associated with endometrial receptivity. Further, senescent cells prevent differentiation of endometrial mesenchymal stem cells in decidualizing cultures. As the cycle progresses, IL-15 activated uterine natural killer (uNK) cells selectively target and clear senescent decidual cells through granule exocytosis. Our findings reveal that acute decidual senescence governs endometrial rejuvenation and remodeling at embryo implantation, and suggest a critical role for uNK cells in maintaining homeostasis in cycling endometrium.
Rapid progress in perinatal care in recent decades has led to a dramatic decline in perinatal, neonatal, and maternal mortality (excluding suicides), and achieved remarkable improvements in ...obstetrical outcomes in Japan. However, while maternal mortality had been on a continuous and steady decline up until 2007 (3.1/100,000 total births), the rate has been fluctuating since then (e.g., 2.7/100,000 in 2014, 3.4/100,000 in 2016). This is likely attributed to a variety of factors that have emerged in the past 20 years due to changes in the environment and social situation surrounding women, such as later marriage and rise in maternal age.In Western countries, “late maternal deaths” occurring between 42 days and one year after delivery are considered to be just as important as “maternal deaths,” i.e., deaths during pregnancy or within 42 days of termination of pregnancy. In particular, suicides attributable to psychiatric disorders have become a serious issue among women less than one year postpartum. However, in Japan, the actual number of deaths by suicide is unknown, since neither death certificates nor postmortem certificates include information on pregnancy and delivery. Despite the fact that the total number of suicide deaths in Japan is known, whether such deaths are associated with perinatal mental issues or not is unclear, and thus, no measures have been taken. Untreated perinatal depression and psychiatric disorders not only cause issues such as suicide, but are also related to pediatric developmental and mental disorders, neglect, and/or child abuse due to impaired nurturing ability. Suicide rates among pregnant and parturient women in Osaka, Tokyo, and Mie are much higher than those of the UK, the US, and Sweden. There is an urgent need to establish a regional support system that facilitates interactions among the obstetrical, pediatric, psychiatric field, and local administrations for monitoring and supporting mothers and infants, as well as a system that allows families, schools, and society to support young people, in order to realize improved preconception health care.
Type VI CRISPR-Cas13 effector enzymes catalyze RNA-guided RNA cleavage and have been harnessed for various technologies, such as RNA detection, targeting, and editing. Recent studies identified ...Cas13bt3 (also known as Cas13X.1) as a miniature Cas13 enzyme, which can be used for knockdown and editing of target transcripts in mammalian cells. However, the action mechanism of the compact Cas13bt3 remains unknown. Here, we report the structures of the Cas13bt3-guide RNA complex and the Cas13bt3-guide RNA-target RNA complex. The structures revealed how Cas13bt3 recognizes the guide RNA and its target RNA and provided insights into the activation mechanism of Cas13bt3, which is distinct from those of the other Cas13a/d enzymes. Furthermore, we rationally engineered enhanced Cas13bt3 variants and ultracompact RNA base editors. Overall, this study improves our mechanistic understanding of the CRISPR-Cas13 enzymes and paves the way for the development of efficient Cas13-mediated transcriptome modulation technologies.
SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) ...vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.
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•Cryo-EM structures of compact AsCas12f in complex with sgRNA and target DNA•AsCas12f engineering by structural analysis and deep mutational scanning methods•Activities of enAsCas12f variants, SpCas9, and AsCas12a variants are comparable•enAsCas12f effectors have been harnessed as a compact transcriptional activation tool
A high-throughput approach, combining deep mutational scanning and structure-informed design, generated miniature AsCas12f variants with enhanced activity, enabling efficient knock-in/knock-out activities and transcriptional activation in mice using an all-in-one AAV vector.
Recent advance in cancer research sheds light on the contribution of mitochondrial respiration in tumorigenesis, as they efficiently produce ATP and oncogenic metabolites that will facilitate cancer ...cell growth. Here we show that a stabilizing factor for mitochondrial supercomplex assembly, COX7RP/COX7A2L/SCAF1, is abundantly expressed in clinical breast and endometrial cancers. Moreover, COX7RP overexpression associates with prognosis of breast cancer patients. We demonstrate that COX7RP overexpression in breast and endometrial cancer cells promotes in vitro and in vivo growth, stabilizes mitochondrial supercomplex assembly even in hypoxic states, and increases hypoxia tolerance. Metabolomic analyses reveal that COX7RP overexpression modulates the metabolic profile of cancer cells, particularly the steady-state levels of tricarboxylic acid cycle intermediates. Notably, silencing of each subunit of the 2-oxoglutarate dehydrogenase complex decreases the COX7RP-stimulated cancer cell growth. Our results indicate that COX7RP is a growth-regulatory factor for breast and endometrial cancer cells by regulating metabolic pathways and energy production.
Maternal mortality rates in Japan declined steadily until around 2007, after which they plateaued, with current numbers residing at 2.7–4.8/100,000 total births. A more accurate grasp on the number ...of maternal deaths and suicide cases among pregnant women and those within 1 year postpartum is needed. These data must be analyzed in order to provide countermeasures and knowledge on emergency life-saving measures. To this end, the entire medical team should attend training so that the various problems may be resolved among multidisciplinary professionals and better treatment and management may be provided, facilitating a proper response to any situation. Establishing cooperative relationships with higher level medical institutions to which patients may need to be transferred is also important. Sharing case histories and courses in regular case conferences and debriefing sessions would also be beneficial.
During the human in vitro fertilization procedure in the assisted reproductive technology, intracytoplasmic sperm injection is routinely used to inject a spermatozoon or a less mature elongating ...spermatid into the oocyte. In some infertile men, round spermatids (haploid male germ cells that have completed meiosis) are the most mature cells visible during testicular biopsy. The microsurgical injection of a round spermatid into an oocyte as a substitute is commonly referred to as round spermatid injection (ROSI). Currently, human ROSI is considered a very inefficient procedure and of no clinical value. Herein, we report the birth and development of 14 children born to 12 women following ROSI of 734 oocytes previously activated by an electric current. The round spermatids came from men who had been diagnosed as not having spermatozoa or elongated spermatids by andrologists at other hospitals after a first Micro-TESE. A key to our success was our ability to identify round spermatids accurately before oocyte injection. As of today, all children born after ROSI in our clinic are without any unusual physical, mental, or epigenetic problems. Thus, for men whose germ cells are unable to develop beyond the round spermatid stage, ROSI can, as a last resort, enable them to have their own genetic offspring.
Endocrine therapy using antiestrogens and aromatase inhibitors is usually efficient to treat patients with hormone-sensitive breast cancer. Many patients with endocrine therapy, however, often ...acquire resistance. In the present study, we performed functional screening using short hairpin RNA library to dissect genes involved in antiestrogen tamoxifen resistance in MCF-7 breast cancer cells. We identified seven candidate genes that are associated with poor prognosis of breast cancer patients based on clinical dataset. The expression levels of six out of seven genes were higher in 4-hydroxytamoxifen (OHT) resistant MCF-7 (OHTR) cells compared with parental MCF-7 cells. Among the six selected genes, siRNA-mediated knockdown of PSMD1 and TSPAN12 markedly reduced the proliferation of OHTR cells. Notably, the knockdown of proteasome 26S subunit PSMD1 exhibited cell cycle arrest and the accumulation of p53 protein through inhibiting p53 protein degradation. In accordance with p53 accumulation, its target genes p21 and SFN were also upregulated by PSMD1 silencing. Taken together, PSMD1 was identified as a potential gene that plays a role in the development of tamoxifen resistance in breast cancer cells. These findings will provide a new insight for the mechanism underlying endocrine therapy resistance and a prognostic and therapeutic molecular target for advanced breast cancer.
Abstract
Endometrial cancer (EC) is a common gynecological malignancy with relatively favourable prognosis, although alternative diagnostic and therapeutic options remain to be explored for advanced ...disease. Recent studies enabled to apply microRNAs (miRs) to clinical cancer management as promising diagnostic and therapeutic biomarkers. We here aimed to identify proliferation-associated miRNAs and characterize their functions in EC cells. Our small RNA-sequencing analysis showed that miR-191 is abundantly expressed in HEC-1A and Ishikawa EC cells along with the high expression of miR-182, which was previously characterized as an EC proliferation-related miRNA in EC. We showed that miR-191 was upregulated in EC tissues than in adjacent normal tissues and its knockdown repressed EC cell proliferation. In silico miRNA target screening identified that ten–eleven translocation 1 (TET1) is one of the putative miR-191 targets. TET1 expression could be downregulated by miR-191 through the mRNA–miRNA interaction in the 3′-untranslated region of TET1. In line with TET1 functions as a methylcytosine dioxygenase, which removes genome-wide DNA methylation marks, decreased TET1 expression resulted in hypermethylation in the promotor region of tumour suppressor adenomatous polyposis coli. Taken together, miR-191 could function as an oncogenic miRNA in EC and serve as a prospective diagnostic and therapeutic target for advanced disease.
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