Genome editing in mammalian cells using the CRISPR type I-D nuclease

Adoption of CRISPR–Cas systems, such as CRISPR–Cas9 and CRISPR–Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I—the most abundant CRISPR system in bacteria—remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense (‘Cascade’: Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR–Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR–Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR–Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells

RNA structure-wide discovery of functional interactions with multiplexed RNA motif library

RNA構造のライブラリ化を通じてRNA 構造ごとにおけるRNA-タンパク質相互作用を大規模に解析するシステム「FOREST」の開発 --RNAを標的とする創薬に道--. 京都大学プレスリリース. 2020-12-09.Biochemical assays and computational analyses have discovered RNA structures throughout various transcripts. However, the roles of these structures are mostly unknown. Here we develop folded RNA element profiling with structure library (FOREST), a multiplexed affinity assay system to identify functional interactions from transcriptome-wide RNA structure datasets. We generate an RNA structure library by extracting validated or predicted RNA motifs from gene-annotated RNA regions. The RNA structure library with an affinity enrichment assay allows for the comprehensive identification of target-binding RNA sequences and structures in a high-throughput manner. As a proof-of-concept, FOREST discovers multiple RNA-protein interaction networks with quantitative scores, including translational regulatory elements that function in living cells. Moreover, FOREST reveals different binding landscapes of RNA G-quadruplex (rG4) structures-binding proteins and discovers rG4 structures in the terminal loops of precursor microRNAs. Overall, FOREST serves as a versatile platform to investigate RNA structure-function relationships on a large scale

Efficacy and Complications of Emergent Transcatheter Arterial Embolization for the Management of Intractable Uterine Bleeding

Objective：Transcatheter arterial embolization（TAE）, including uterine artery embolization（UAE）, is effective for the management of obstetric and gynecologic hemorrhage. Some adverse effects have been reported with TAE, such as amenorrhea, endometrial trauma, and subsequent infertility. Herein we report the efficacy and complications of emergent TAE for the management of severe intractable uterine bleeding at our institute.Methods：From 2010 to 2019, thirty-eight patients underwent emergent TAE for intractable uterine bleeding. We evaluated the efficacy and complications of TAE, including a change in menstruation, fertility, and pregnancy outcomes in perinatal patients（group A；n＝23）, and in patients with gynecologic hemorrhage（group B；n＝15）.Results：In group A, 7 cases of retained placenta, 4 cases of postpartum hemorrhage, 2 cases of placenta accrete, 2 cases of uterine artery pseudoaneurysm, 2 cases of cervical pregnancy, 1 case of cesarean scar pregnancy, and 5 cases of unexplained hemorrhage were included. The median age of the group A was 37. In group B, 4 cases of uterine artery pseudoaneurysm, 2 cases of uterine arteriovenous malformation, 3 cases of uterine fibroids, 1case of adenomyosis, and 5 cases of unexplained hemorrhage were included. The median age of the group B was 39. The first attempt at TAE successfully controlled hemorrhage in 33 of 38 patients （86.8％）without major complications, and the remaining 5 patients required an additional attempt at TAE to control hemorrhage. One patient（2.6％）had transient buttock pain and foot ischemia. Among the 33 patients who had adequate follow-up care, all patients resumed regular menstruation. The median time to resume regular menstruation after TAE was 3 months （range, 1-13 months）in group A（n＝20）and 1 month（range, 1-6 months）in group B（n＝13）. Four of patients had 6 pregnancies in total：3 full-term live births, 2 missed abortions, and 1 artificial abortion. Among the 13 patients who desired pregnancy, 3（23％）conceived spontaneously.Conclusions：This retrospective study showed that emergent TAE may be effective and safe in treating intractable uterine bleeding with a high success rate. Ovarian and endometrial function were preserved based on the relatively early return of menstruation. Further prospective investigations with large number of patients are needed to confirm the preservation of ovarian function, fertility, and pregnancy outcome in reproductive-aged women

Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires a Bicc1-Ccr4 RNA degradation complex

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3′ untranslated region (3′-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3′-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3′-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node

Long-Term Memory Engram Cells Are Established by c-Fos/CREB Transcriptional Cycling

Summary: Training-dependent increases in c-fos have been used to identify engram cells encoding long-term memories (LTMs). However, the interaction between transcription factors required for LTM, including CREB and c-Fos, and activating kinases such as phosphorylated ERK (pERK) in the establishment of memory engrams has been unclear. Formation of LTM of an aversive olfactory association in flies requires repeated training trials with rest intervals between trainings. Here, we find that prolonged rest interval-dependent increases in pERK induce transcriptional cycling between c-Fos and CREB in a subset of KCs in the mushroom bodies, where olfactory associations are made and stored. Preexisting CREB is required for initial c-fos induction, while c-Fos is required later to increase CREB expression. Blocking or activating c-fos-positive engram neurons inhibits memory recall or induces memory-associated behaviors. Our results suggest that c-Fos/CREB cycling defines LTM engram cells required for LTM. : Long-term memory (LTM) requires transcription factors, including CREB and c-Fos. Miyashita et al. show that spaced training, which induces LTM, activates c-Fos/CREB cycling, where increases in c-Fos require CREB and increases in CREB require c-Fos. c-Fos/CREB cycling defines LTM engram cells, and modulating the activity of these cells alters memory-associated behaviors. Keywords: long-term memory, CREB, c-fos, spacing effect, MAPK, Drosophila, memory engram, transcriptional cyclin

Cytogenetically Unrelated Clones in Acute Myeloid Leukemia Showing Different Responses to Chemotherapy

We report a case of acute myeloid leukemia (AML) with two cytogenetically unrelated clones. The patient was a 45-year-old male who was diagnosed with acute monoblastic leukemia (AMoL). Initial G-band analysis showed 51,XY,+6,+8,inv(9)(p12q13)c,+11,+13,+19[12]/52,idem,+Y[8], but G-band analysis after induction therapy showed 45,XY,-7,inv(9)(p12q13)c[19]/46,XY,inv(9)(p12q13)c[1]. Retrospective FISH analysis revealed a cryptic monosomy 7 clone in the initial AML sample. The clone with multiple trisomies was eliminated after induction therapy and never recurred, but a clone with monosomy 7 was still detected in myelodysplastic marrow with a normal blast percentage. Both clones were successfully eliminated after related peripheral blood stem cell transplantation, but the patient died of relapsed AML with monosomy 7. We concluded that one clone was de novo AMoL with chromosome 6, 8, 11, 13, and 19 trisomy and that the other was acute myeloid leukemia with myelodysplasia-related changes　(AML-MRC) with chromosome 7 monosomy showing different responses to chemotherapy. Simultaneous onset of cytogenetically unrelated hematological malignancies that each have a different disease status is a rare phenomenon but is important to diagnose for a correct understanding of the disease status and for establishing an appropriate treatment strategy