Gut microbiota contributes to high-altitude hypoxia acclimatization of human populations.

BACKGROUND: The relationship between human gut microbiota and high-altitude hypoxia acclimatization remains highly controversial. This stems primarily from uncertainties regarding both the potential temporal changes in the microbiota under such conditions and the existence of any dominant or core bacteria that may assist in host acclimatization. RESULTS: To address these issues, and to control for variables commonly present in previous studies which significantly impact the results obtained, namely genetic background, ethnicity, lifestyle, and diet, we conducted a 108-day longitudinal study on the same cohort comprising 45 healthy Han adults who traveled from lowland Chongqing, 243 masl, to high-altitude plateau Lhasa, Xizang, 3658 masl, and back. Using shotgun metagenomic profiling, we study temporal changes in gut microbiota composition at different timepoints. The results show a significant reduction in the species and functional diversity of the gut microbiota, along with a marked increase in functional redundancy. These changes are primarily driven by the overgrowth of Blautia A, a genus that is also abundant in six independent Han cohorts with long-term duration in lower hypoxia environment in Shigatse, Xizang, at 4700 masl. Further animal experiments indicate that Blautia A-fed mice exhibit enhanced intestinal health and a better acclimatization phenotype to sustained hypoxic stress. CONCLUSIONS: Our study underscores the importance of Blautia A species in the gut microbiota's rapid response to high-altitude hypoxia and its potential role in maintaining intestinal health and aiding host adaptation to extreme environments, likely via anti-inflammation and intestinal barrier protection.

Methylation entropy landscape of Chinese long-lived individuals reveals lower epigenetic noise related to human healthy aging.

The transition from ordered to noisy is a significant epigenetic signature of aging and age-related disease. As a paradigm of healthy human aging and longevity, long-lived individuals (LLI, >90 years old) may possess characteristic strategies in coping with the disordered epigenetic regulation. In this study, we constructed high-resolution blood epigenetic noise landscapes for this cohort by a methylation entropy (ME) method using whole genome bisulfite sequencing (WGBS). Although a universal increase in global ME occurred with chronological age in general control samples, this trend was suppressed in LLIs. Importantly, we identified 38,923 genomic regions with LLI-specific lower ME (LLI-specific lower entropy regions, for short, LLI-specific LERs). These regions were overrepresented in promoters, which likely function in transcriptional noise suppression. Genes associated with LLI-specific LERs have a considerable impact on SNP-based heritability of some aging-related disorders (e.g., asthma and stroke). Furthermore, neutrophil was identified as the primary cell type sustaining LLI-specific LERs. Our results highlight the stability of epigenetic order in promoters of genes involved with aging and age-related disorders within LLI epigenomes. This unique epigenetic feature reveals a previously unknown role of epigenetic order maintenance in specific genomic regions of LLIs, which helps open a new avenue on the epigenetic regulation mechanism in human healthy aging and longevity.

CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression.

Metastatic prostate cancer (PCa) poses a significant therapeutic challenge with high mortality rates. Utilizing CRISPR-Cas9 in vivo, we target five potential tumor suppressor genes (Pten, Trp53, Rb1, Stk11, and RnaseL) in the mouse prostate, reaching humane endpoint after eight weeks without metastasis. By further depleting three epigenetic factors (Kmt2c, Kmt2d, and Zbtb16), lung metastases are present in all mice. While whole genome sequencing reveals few mutations in coding sequence, RNA sequencing shows significant dysregulation, especially in a conserved genomic region at chr5qE1 regulated by KMT2C. Depleting Odam and Cabs1 in this region prevents metastasis. Notably, the gene expression signatures, resulting from our study, predict progression-free and overall survival and distinguish primary and metastatic human prostate cancer. This study emphasizes positive genetic interactions between classical tumor suppressor genes and epigenetic modulators in metastatic PCa progression, offering insights into potential treatments.

Scrutiny of genome-wide somatic mutation profiles in centenarians identifies the key genomic regions for human longevity.

Somatic mutations accumulate with age and are associated closely with human health, their characterization in longevity cohorts remains largely unknown. Here, by analyzing whole genome somatic mutation profiles in 73 centenarians and 51 younger controls in China, we found that centenarian genomes are characterized by a markedly skewed distribution of somatic mutations, with many genomic regions being specifically conserved but displaying a high function potential. This, together with the observed more efficient DNA repair ability in the long-lived individuals, supports the existence of key genomic regions for human survival during aging, with their integrity being of essential to human longevity.

ER stress induces caspase-2-tBID-GSDME-dependent cell death in neurons lytically infected with herpes simplex virus type 2.

Neurotropic viruses, including herpes simplex virus (HSV) types 1 and 2, have the capacity to infect neurons and can cause severe diseases. This is associated with neuronal cell death, which may contribute to morbidity or even mortality if the infection is not controlled. However, the mechanistic details of HSV-induced neuronal cell death remain enigmatic. Here, we report that lytic HSV-2 infection of human neuron-like SH-SY5Y cells and primary human and murine brain cells leads to cell death mediated by gasdermin E (GSDME). HSV-2-induced GSDME-mediated cell death occurs downstream of replication-induced endoplasmic reticulum stress driven by inositol-requiring kinase 1α (IRE1α), leading to activation of caspase-2, cleavage of the pro-apoptotic protein BH3-interacting domain death agonist (BID), and mitochondria-dependent activation of caspase-3. Finally, necrotic neurons released alarmins, which activated inflammatory responses in human iPSC-derived microglia. In conclusion, lytic HSV infection in neurons activates an ER stress-driven pathway to execute GSDME-mediated cell death and promote inflammation.

Dual intron-targeted CRISPR-Cas9-mediated disruption of the AML RUNX1-RUNX1T1 fusion gene effectively inhibits proliferation and decreases tumor volume in vitro and in vivo.

Oncogenic fusion drivers are common in hematological cancers and are thus relevant targets of future CRISPR-Cas9-based treatment strategies. However, breakpoint-location variation in patients pose a challenge to traditional breakpoint-targeting CRISPR-Cas9-mediated disruption strategies. Here we present a new dual intron-targeting CRISPR-Cas9 treatment strategy, for targeting t(8;21) found in 5-10% of de novo acute myeloid leukemia (AML), which efficiently disrupts fusion genes without prior identification of breakpoint location. We show in vitro growth rate and proliferation reduction by 69 and 94% in AML t(8;21) Kasumi-1 cells, following dual intron-targeted disruption of RUNX1-RUNX1T1 compared to a non t(8;21) AML control. Furthermore, mice injected with RUNX1-RUNX1T1-disrupted Kasumi-1 cells had in vivo tumor growth reduction by 69 and 91% compared to controls. Demonstrating the feasibility of RUNX1-RUNX1T1 disruption, these findings were substantiated in isolated primary cells from a patient diagnosed with AML t(8;21). In conclusion, we demonstrate proof-of-principle of a dual intron-targeting CRISPR-Cas9 treatment strategy in AML t(8;21) without need for precise knowledge of the breakpoint location.

Mitogenome evidence shows two radiation events and dispersals of matrilineal ancestry from northern coastal China to the Americas and Japan.

Although it is widely recognized that the ancestors of Native Americans (NAs) primarily came from Siberia, the link between mitochondrial DNA (mtDNA) lineage D4h3a (typical of NAs) and D4h3b (found so far only in East China and Thailand) raises the possibility that the ancestral sources for early NAs were more variegated than hypothesized. Here, we analyze 216 contemporary (including 106 newly sequenced) D4h mitogenomes and 39 previously reported ancient D4h data. The results reveal two radiation events of D4h in northern coastal China, one during the Last Glacial Maximum and the other within the last deglaciation, which facilitated the dispersals of D4h sub-branches to different areas including the Americas and the Japanese archipelago. The coastal distributions of the NA (D4h3a) and Japanese lineages (D4h1a and D4h2), in combination with the Paleolithic archaeological similarities among Northern China, the Americas, and Japan, lend support to the coastal dispersal scenario of early NAs.

Impact of U2AF1 mutations on circular RNA expression in myelodysplastic neoplasms.

Mutations in U2AF1 are relatively common in myelodysplastic neoplasms (MDS) and are associated with an inferior prognosis, but the molecular mechanisms underlying this are not fully elucidated. Circular RNAs (circRNAs) have been implicated in cancer, but it is unknown how mutations in splicing factors may impact on circRNA biogenesis. Here, we used RNA-sequencing to investigate the effects of U2AF1 mutations on circRNA expression in K562 cells with a doxycycline-inducible U2AF1S34 mutation, in a mouse model with a doxycycline-inducible U2AF1S34 mutation, and in FACS-sorted CD34+ bone marrow cells from MDS patients with either U2AF1S34 or U2AF1Q157 mutations. In all contexts, we found an increase in global circRNA levels in the U2AF1-mutated setting, which was independent of expression changes in the cognate linear host genes. In patients, the U2AF1S34 and U2AF1Q157 mutations were both associated with an overall increased expression of circRNAs. circRNAs generated by a non-Alu-mediated mechanism generally showed the largest increase in expression levels. Several well-described cancer-associated circRNAs, including circZNF609 and circCSNK1G3, were upregulated in MDS patients with U2AF1 mutations compared to U2AF1-wildtype MDS controls. In conclusion, high circRNA expression is observed in association with U2AF1 mutations in three biological systems, presenting an interesting possibility for biomarker and therapeutic investigation.

A truncated reverse transcriptase enhances prime editing by split AAV vectors.

Prime editing is a new CRISPR-based, genome-editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with adeno-associated virus (AAV) viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4-fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. We also show that this minimized PE gives rise to superior lentiviral vector titers (46-fold) over the regular PE in an all-in-one PE lentiviral vector. We finally deliver the minimized PE to mouse liver by dual AAV8 vectors and show up to 6% precise editing of the PCSK9 gene, thereby demonstrating the value of this truncated split PE system for in vivo applications.

Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi.

Targeted transcriptional activation or interference can be induced with the CRISPR-Cas9 system (CRISPRa/CRISPRi) using nuclease-deactivated Cas9 fused to transcriptional effector molecules. These technologies have been used in cancer cell lines, particularly for genome-wide functional genetic screens using lentiviral vectors. However, CRISPRa and CRISPRi have not yet been widely applied to ex vivo cultured primary cells with therapeutic relevance owing to a lack of effective and nontoxic delivery modalities. Here we develop CRISPRa and CRISPRi platforms based on RNA or ribonucleoprotein (RNP) delivery by electroporation and show transient, programmable gene regulation in primary cells, including human CD34+ hematopoietic stem and progenitor cells (HSPCs) and human CD3+ T cells. We show multiplex and orthogonal gene modulation using multiple sgRNAs and CRISPR systems from different bacterial species, and we show that CRISPRa can be applied to manipulate differentiation trajectories of HSPCs. These platforms constitute simple and effective means to transiently control transcription and are easily adopted and reprogrammed to new target genes by synthetic sgRNAs. We believe these technologies will find wide use in engineering the transcriptome for studies of stem cell biology and gene function, and we foresee that they will be implemented to develop and enhance cellular therapeutics.

Complete mitogenomes document substantial genetic contribution from the Eurasian Steppe into northern Pakistani Indo-Iranian speakers.

To elucidate whether Bronze Age population dispersals from the Eurasian Steppe to South Asia contributed to the gene pool of Indo-Iranian-speaking groups, we analyzed 19,568 mitochondrial DNA (mtDNA) sequences from northern Pakistani and surrounding populations, including 213 newly generated mitochondrial genomes (mitogenomes) from Iranian and Dardic groups, both speakers from the ancient Indo-Iranian branch in northern Pakistan. Our results showed that 23% of mtDNA lineages with west Eurasian origin arose in situ in northern Pakistan since ~5000 years ago (kya), a time depth very close to the documented Indo-European dispersals into South Asia during the Bronze Age. Together with ancient mitogenomes from western Eurasia since the Neolithic, we identified five haplogroups (~8.4% of maternal gene pool) with roots in the Steppe region and subbranches arising (age ~5-2 kya old) in northern Pakistan as genetic legacies of Indo-Iranian speakers. Some of these haplogroups, such as W3a1b that have been found in the ancient samples from the late Bronze Age to the Iron Age period individuals of Swat Valley northern Pakistan, even have sub-lineages (age ~4 kya old) in the southern subcontinent, consistent with the southward spread of Indo-Iranian languages. By showing that substantial genetic components of Indo-Iranian speakers in northern Pakistan can be traced to Bronze Age in the Steppe region, our study suggests a demographic link with the spread of Indo-Iranian languages, and further highlights the corridor role of northern Pakistan in the southward dispersal of Indo-Iranian-speaking groups.

Design and Evaluation of Guide RNA Transcripts with a 3'-Terminal HDV Ribozyme to Enhance CRISPR-Based Gene Inactivation.

The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system, now reclassified as Cas12a, is a DNA-editing platform analogous to the widely used CRISPR-Cas9 system. The Cas12a system exhibits several distinct features over the CRISPR-Cas9 system, such as increased specificity and a smaller gene size to encode the nuclease and the matching CRISPR guide RNA (crRNA), which could mitigate off-target and delivery problems, respectively, described for the Cas9 system. However, the Cas12a system exhibits reduced gene editing efficiency compared to Cas9. A closer inspection of the crRNA sequence raised some uncertainty about the actual 5' and 3'-ends. RNA Polymerase (Pol) III promoters are generally used for the production of small RNAs with a precise 5' terminus, but the Pol III enzyme generates small RNAs with 3' U-tails of variable length. To optimize the CRISPR-Cas12a system, we describe the inclusion of a self-cleaving ribozyme in the vector design to facilitate accurate 3'-end processing of the crRNA transcript to produce precise molecules. This optimized design enhanced not only the gene editing efficiency, but also the activity of the catalytically inactive Cas12a-based CRISPR gene activation platform. We thus generated an improved CRISPR-Cas12a system for more efficient gene editing and gene regulation purposes.

Engineered miniature H1 promoters with dedicated RNA polymerase II or III activity.

RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both).

Author Correction: STEEP mediates STING ER exit and activation of signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

STEEP mediates STING ER exit and activation of signaling.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.

Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA.

The CRISPR-Cas9 system has been used for genome editing of various organisms. We reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections with a single guide RNA (gRNA) and subsequent viral escape, but complete inactivation of infectious HIV with certain combinations of two gRNAs. The new RNA-guided endonuclease system CRISPR-Cas12a (formerly Cpf1) may provide a more promising tool for genome engineering with increased activity and specificity. We compared Cas12a to the original Cas9 system for inactivation of the integrated HIV DNA genome. Superior antiviral activity is reported for Cas12a, which can achieve full HIV inactivation with only a single gRNA (called crRNA). We propose that the different architecture of Cas9 versus Cas12a endonuclease explains this effect. We also disclose that DNA cleavage by the Cas12a endonuclease and subsequent DNA repair causes mutations with a sequence profile that is distinct from that of Cas9. Both CRISPR systems can induce the typical small deletions around the site of DNA cleavage and subsequent repair, but Cas12a does not induce the pure DNA insertions that are routinely observed for Cas9. Although these typical signatures are apparent in many literature studies, this is the first report that documents these striking differences.

CRISPR therapy towards an HIV cure.

Tools based on RNA interference (RNAi) and the recently developed clustered regularly short palindromic repeats (CRISPR) system enable the selective modification of gene expression, which also makes them attractive therapeutic reagents for combating HIV infection and other infectious diseases. Several parallels can be drawn between the RNAi and CRISPR-Cas9 platforms. An ideal RNAi or CRISPR-Cas9 therapeutic strategy for treating infectious or genetic diseases should exhibit potency, high specificity and safety. However, therapeutic applications of RNAi and CRISPR-Cas9 have been challenged by several major limitations, some of which can be overcome by optimal design of the therapy or the design of improved reagents. In this review, we will discuss some advantages and limitations of anti-HIV strategies based on RNAi and CRISPR-Cas9 with a focus on the efficiency, specificity, off-target effects and delivery methods.

Influence of a 3' Terminal Ribozyme on AgoshRNA Biogenesis and Activity.

Short hairpin RNAs (shRNAs) can induce gene silencing via the RNA interference (RNAi) mechanism. We designed an alternative shRNA molecule with a relatively short base-paired stem that bypasses Dicer and instead is processed by the Argonaute 2 (Ago2) protein into a single guide RNA strand that effectively induces RNAi. We called these molecules AgoshRNAs. Active anti-HIV AgoshRNAs were developed, but their RNAi activity was generally reduced compared with the matching shRNAs. In an attempt to further optimize the AgoshRNA design, we inserted several self-cleaving ribozymes at the 3' terminus of the transcribed AgoshRNA and evaluated the impact on AgoshRNA processing and activity. The hepatitis delta virus (HDV) ribozyme is efficiently removed from the transcribed AgoshRNAs and generates a uniform 3' overhang, which translates into the enhanced antiviral activity of these molecules.

Boosting AgoshRNA activity by optimized 5'-terminal nucleotide selection.

RNA interference (RNAi) can be triggered by synthetic small interfering RNAs (siRNAs) or transgene-expressed short hairpin RNAs (shRNAs). Recent evidence indicates that shRNA molecules, with a relatively short stem and small loop, are processed by Argonaute 2 protein (Ago2). We named these molecules AgoshRNA as Ago2 is involved in both the processing and the subsequent mRNA-silencing reaction. This alternative processing route yields only a single guide strand, which thus avoids potential off-target effects induced by the passenger strand of a regular shRNA. We recently described that the introduction of a 5'-terminal purine (A or G) and a mismatch at the bottom of the hairpin enhances the AgoshRNA activity. The critical 5'-terminal nucleotide (nt) represents the +1 position of the transcriptional promoter, which influences the transcriptional efficiency and initiation accuracy as demonstrated for the H1 RNA polymerase (Pol) III promoter. These findings highlight the necessity of considering Pol III requirements in the design of optimized AgoshRNA cassettes. In this study, we report the design and expression of potent AgoshRNAs by two other popular Pol III promoters: U6 and 7SK, which were recently reported to have a distinct transcription profile compared to the H1 promoter. We propose general rules for the design and expression of potent AgoshRNA molecules using Pol III cassettes, which should augment the application of novel AgoshRNA reagents for basic research and therapeutic purposes.

A Single H1 Promoter Can Drive Both Guide RNA and Endonuclease Expression in the CRISPR-Cas9 System.

The RNA-guided endonuclease Cas9 (CRISPR-Cas9) genome editing system has been widely used for biomedical research and holds great potential for therapeutic applications in eukaryotes. The conventional vector-based CRISPR-Cas9 delivery system requires two different RNA polymerase promoters for expression of the guide RNA (gRNA) and Cas9 endonuclease. The large size and relative complexity of such CRISPR transgene cassettes impede their broad implementation, especially in gene therapy applications with viral vectors that have a limited packaging capacity. Here, we report the design of a single-promoter-driven CRISPR-Cas9 system that uses the dual-polymerase (Pol II and Pol III) activity of the H1 promoter. This size reduction strategy of the vector insert provides a significant titer advantage in the lentiviral vector over the regular CRISPR system.