In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates.

Gene-editing technologies, which include the CRISPR-Cas nucleases1-3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a 'once-and-done' approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs.

Distinct functions and temporal regulation of methylated histone H3 during early embryogenesis.

During the first hours of embryogenesis, formation of higher-order heterochromatin coincides with the loss of developmental potential. Here, we examine the relationship between these two events, and we probe the processes that contribute to the timing of their onset. Mutations that disrupt histone H3 lysine 9 (H3K9) methyltransferases reveal that the methyltransferase MET-2 helps terminate developmental plasticity, through mono- and di-methylation of H3K9 (me1/me2), and promotes heterochromatin formation, through H3K9me3. Although loss of H3K9me3 perturbs formation of higher-order heterochromatin, embryos are still able to terminate plasticity, indicating that the two processes can be uncoupled. Methylated H3K9 appears gradually in developing C. elegans embryos and depends on nuclear localization of MET-2. We find that the timing of H3K9me2 and nuclear MET-2 is sensitive to rapid cell cycles, but not to zygotic genome activation or cell counting. These data reveal distinct roles for different H3K9 methylation states in the generation of heterochromatin and loss of developmental plasticity by MET-2, and identify the cell cycle as a crucial parameter of MET-2 regulation.

Cracking the cytotoxicity code: apoptotic induction of 10-acetylirciformonin B is mediated through ROS generation and mitochondrial dysfunction.

A marine furanoterpenoid derivative, 10-acetylirciformonin B (10AB), was found to inhibit the proliferation of leukemia, hepatoma, and colon cancer cell lines, with selective and significant potency against leukemia cells. It induced DNA damage and apoptosis in leukemia HL 60 cells. To fully understand the mechanism behind the 10AB apoptotic induction against HL 60 cells, we extended our previous findings and further explored the precise molecular targets of 10AB. We found that the use of 10AB increased apoptosis by 8.9%-87.6% and caused disruption of mitochondrial membrane potential (MMP) by 15.2%-95.2% in a dose-dependent manner, as demonstrated by annexin-V/PI and JC-1 staining assays, respectively. Moreover, our findings indicated that the pretreatment of HL 60 cells with N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger, diminished MMP disruption and apoptosis induced by 10AB, suggesting that ROS overproduction plays a crucial rule in the cytotoxic activity of 10AB. The results of a cell-free system assay indicated that 10AB could act as a topoisomerase catalytic inhibitor through the inhibition of topoisomerase IIα. On the protein level, the expression of the anti-apoptotic proteins Bcl-xL and Bcl-2, caspase inhibitors XIAP and survivin, as well as hexokinase II were inhibited by the use of 10AB. On the other hand, the expression of the pro-apoptotic protein Bax was increased after 10AB treatment. Taken together, our results suggest that 10AB-induced apoptosis is mediated through the overproduction of ROS and the disruption of mitochondrial metabolism.

10-acetylirciformonin B, a sponge furanoterpenoid, induces DNA damage and apoptosis in leukemia cells.

10-Acetylirciformonin B, a furanoterpenoid derived from irciformonin B found in a marine sponge, has been reported to possess potent cytotoxic activity against several cancer cell lines. However, the mechanism of its apoptotic activity against human leukemia cells has never been reported. The purpose of this study was to investigate the cytotoxic effects of 10-acetylirciformonin B and its possible mechanism of action against leukemia HL 60 cells. We found that 10-acetylirciformonin B decreased cell viability through the inhibition of cell growth as well as the induction of DNA damage and apoptosis in a dose-dependent manner. The induction of DNA damage was mediated by the increase of p-CHK2 and γ-H2A.X, which was suggested from the increase of tail movement in the neutral Comet assay. Induction of apoptosis was mediated with the increase in caspases 8, 9 and 3 activation as well as PARP cleavage. In summary, our resultsindicate that 10-acetylirciformonin B treatment causes apoptosis in leukaemia cells; probably through a caspase-dependent regulatory pathway.

Molecular characterization of precise in vivo targeted gene integration in human cells using AAVHSC15.

Targeted gene integration via precise homologous recombination (HR)-based gene editing has the potential to correct genetic diseases. AAV (adeno-associated virus) can mediate nuclease-free gene integration at a disease-causing locus. Therapeutic application of AAV gene integration requires quantitative molecular characterization of the edited sequence that overcome technical obstacles such as excess episomal vector genomes and lengthy homology arms. Here we describe a novel molecular methodology that utilizes quantitative next-generation sequencing to characterize AAV-mediated targeted insertion and detects the presence of unintended mutations. The methods described here quantify targeted insertion and query the entirety of the target locus for the presence of insertions, deletions, single nucleotide variants (SNVs) and integration of viral components such as inverted terminal repeats (ITR). Using a humanized liver murine model, we demonstrate that hematopoietic stem-cell derived AAVHSC15 mediates in vivo targeted gene integration into human chromosome 12 at the PAH (phenylalanine hydroxylase) locus at 6% frequency, with no sign of co-incident random mutations at or above a lower limit of detection of 0.5% and no ITR sequences at the integration sites. Furthermore, analysis of heterozygous variants across the targeted locus using the methods described shows a pattern of strand cross-over, supportive of an HR mechanism of gene integration with similar efficiencies across two different haplotypes. Rapid advances in the application of AAV-mediated nuclease-free target integration, or gene editing, as a new therapeutic modality requires precise understanding of the efficiency and the nature of the changes being introduced to the target genome at the molecular level. This work provides a framework to be applied to homologous recombination gene editing platforms for assessment of introduced and natural sequence variation across a target site.

Regulated nuclear accumulation of a histone methyltransferase times the onset of heterochromatin formation in C. elegans embryos.

Heterochromatin formation during early embryogenesis is timed precisely, but how this process is regulated remains elusive. We report the discovery of a histone methyltransferase complex whose nuclear accumulation and activation establish the onset of heterochromatin formation in Caenorhabditis elegans embryos. We find that the inception of heterochromatin generation coincides with the accumulation of the histone H3 lysine 9 (H3K9) methyltransferase MET-2 (SETDB) into nuclear hubs. The absence of MET-2 results in delayed and disturbed heterochromatin formation, whereas accelerated nuclear localization of the methyltransferase leads to precocious H3K9 methylation. We identify two factors that bind to and function with MET-2: LIN-65, which resembles activating transcription factor 7-interacting protein (ATF7IP) and localizes MET-2 into nuclear hubs, and ARLE-14, which is orthologous to adenosine 5'-diphosphate-ribosylation factor-like 14 effector protein (ARL14EP) and promotes stable association of MET-2 with chromatin. These data reveal that nuclear accumulation of MET-2 in conjunction with LIN-65 and ARLE-14 regulates timing of heterochromatin domains during embryogenesis.

Resistance enhancement of transgenic tomato to bacterial pathogens by the heterologous expression of sweet pepper ferredoxin-I protein.

ABSTRACT Expression of a foreign gene to enhance plant disease resistance to bacterial pathogens is a favorable strategy. It has been demonstrated that expressing sweet pepper ferredoxin-I protein (PFLP) in transgenic plants can enhance disease resistance to bacterial pathogens that infect leaf tissue. In this study, PFLP was applied to protect tomato (Lycopersicon esculentum cv. cherry Cln1558a) from the root-infecting pathogen, Ralstonia solanacearum. Independent R. solanacearum resistant T(1) lines were selected and bred to produce homozygous T(2) generations. Selected T(2) transgenic lines 24-18-7 and 26-2-1a, which showed high expression levels of PFLP in root tissue, were resistant to disease caused by R. solanacearum. In contrast, the transgenic line 23-17-1b and nontransgenic tomato, which showed low expression levels of PFLP in root tissue, were not resistant to R. solanacearum infection. The expansion of R. solanacearum populations in stem tissue of transgenic tomato line 24-18-7 was limited compared with the nontransgenic tomato Cln1558a. Using a detached leaf assay, transgenic line 24-18-7 was also resistant to maceration caused by E. carotovora subsp. carotovora; however, resistance to E. carotovora subsp. carotovora was less apparent in transgenic lines 26-2-1a and 23-17-1b. These results demonstrate that PFLP is able to enhance disease resistance at different levels to bacterial pathogens in individual tissue of transgenic tomato.

Cloning and characterization of a plant defensin VaD1 from azuki bean.

A recombinant mungbean defensin VrD1 was previously shown to exhibit antifungal and bruchid-resistant activity. To study the function and regulation of VrD1, genomic DNAs of plant defensins were isolated from Vigna radiata VC6089A and azuki bean Vigna angularis Kao Hsiung No. 6. The azuki bean defensin genomic DNA VaD1 was sequenced and converted to VaD1 cDNA. VaD1 defensin was purified from Vigna angularis Kao Hsiung No. 6 to apparent homogeneity. The complete amino acid sequence of the purified VaD1 was determined and was found to be exactly the same as the sequence deduced from VaD1 cDNA. VaD1 is a basic protein containing 46 amino acids with four conserved disulfide bonds and shares high sequence homology (78.3%) with VrD1. VaD1 inhibited the growth of Fusarium oxysporum, Fusarium oxysporum f. sp. pisi, Staphylococcus epidermidis, and Salmonella typhimurium. VaD1 also inhibited in vitro protein synthesis and bruchid larval development, but was less active than the recombinant VrD1.

Lack of type VI adenylyl cyclase (AC6) leads to abnormal sympathetic tone in neonatal mice.

Visceral functions are regulated by a basal sympathetic nerve discharge (SND), also known as 'sympathetic tone'. We demonstrate herein that AC6 existed in tyrosine hydroxylase-positive rostral ventrolateral medulla neurons in the brainstem. Adenylyl cyclase (AC) assays showed lower basal and pituitary adenylate cyclase-activating peptide-evoked AC activities in the brainstem of AC6-null mice, indicating that AC6 is a prominent AC isozyme in the brainstem. Furthermore, two independent lines of AC6-null mice exhibited a much higher SND, recorded from splanchnic sympathetic nerves of neonatal brainstem-spinal cord preparations, than wildtype mice. An assay of urine noradrenaline confirmed this observation. Collectively, AC6 plays a critical role in the regulation of sympathetic tone.

Repression of meiotic genes by antisense transcription and by Fkh2 transcription factor in Schizosaccharomyces pombe.

In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the "unspliced" signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.

A conserved regulatory role for antisense RNA in meiotic gene expression in yeast.

A significant fraction of the eukaryotic genome is transcribed into RNAs that do not encode proteins, termed non-coding RNA (ncRNA). One class of ncRNA that is of particular interest is antisense RNAs, which are complementary to protein coding transcripts (mRNAs). In this article, we summarize recent studies using different yeasts that reveal a conserved pattern in which meiotically expressed genes have antisense transcripts in vegetative cells. These antisense transcripts repress the basal transcription of the mRNA during vegetative growth and are diminished as cells enter meiosis. While the mechanism(s) by which these antisense RNAs interfere with production of sense transcripts is not yet understood, the effects appear to be independent of the canonical RNAi machinery.

The fission yeast RNA binding protein Mmi1 regulates meiotic genes by controlling intron specific splicing and polyadenylation coupled RNA turnover.

The polyA tails of mRNAs are monitored by the exosome as a quality control mechanism. We find that fission yeast, Schizosaccharomyces pombe, adopts this RNA quality control mechanism to regulate a group of 30 or more meiotic genes at the level of both splicing and RNA turnover. In vegetative cells the RNA binding protein Mmi1 binds to the primary transcripts of these genes. We find the novel motif U(U/C/G)AAAC highly over-represented in targets of Mmi1. Mmi1 can specifically regulate the splicing of particular introns in a transcript: it inhibits the splicing of introns that are in the vicinity of putative Mmi1 binding sites, while allowing the splicing of other introns that are far from such sites. In addition, binding of Mmi1, particularly near the 3' end, alters 3' processing to promote extremely long polyA tails of up to a kilobase. The hyperadenylated transcripts are then targeted for degradation by the nuclear exonuclease Rrp6. The nuclear polyA binding protein Pab2 assists this hyperadenylation-mediated RNA decay. Rrp6 also targets other hyperadenylated transcripts, which become hyperadenylated in an unknown, but Mmi1-independent way. Thus, hyperadenylation may be a general signal for RNA degradation. In addition, binding of Mmi1 can affect the efficiency of 3' cleavage. Inactivation of Mmi1 in meiosis allows meiotic expression, through splicing and RNA stabilization, of at least 29 target genes, which are apparently constitutively transcribed.

A novel synthetic protoapigenone analogue, WYC02-9, induces DNA damage and apoptosis in DU145 prostate cancer cells through generation of reactive oxygen species.

The protoapigenone analogue WYC02-9, a novel synthetic flavonoid, has been shown to act against a variety of experimental tumors. However, its effects on prostate cancer and its mechanism of action are unknown. Thus, WYC02-9 was investigated for its cytotoxicity against DU145 prostate cancer cells, as was the underlying mechanisms by which WYC02-9 might induce DNA damage and apoptotic cell death through reactive oxygen species (ROS). WYC02-9 inhibited the cell growth of three prostate cancer cell lines, especially DU145 cells. In DU145 cells, WYC02-9 increased the generation of intracellular ROS, followed by induction of DNA damage and activation of the ATM-p53-H2A.X pathway and checkpoint-related signals Chk1/Chk2, which led to increased numbers of cells in the S and G2/M phases of the cell cycle. Furthermore, WYC02-9 induced apoptotic cell death through mitochondrial membrane potential decrease and activation of caspase-9, caspase-3, and PARP. The above effects were all prevented by the ROS scavenger N-acetylcysteine. Administration of WYC02-9 in a nude mouse DU145 xenograft model further identified the anti-cancer activity of WYC02-9. These findings therefore suggest that WYC02-9-induced DNA damage and mitochondria-dependent cell apoptosis in DU145 cells are mediated via ROS generation.

Comparative functional genomics of the fission yeasts.

The fission yeast clade--comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus, and S. japonicus--occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, which suggests a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the budding yeast of Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.

A complex gene regulatory mechanism that operates at the nexus of multiple RNA processing decisions.

Expression of crs1 pre-mRNA, encoding a meiotic cyclin, is blocked in actively growing fission yeast cells by a multifaceted mechanism. The most striking feature is that in vegetative cells, crs1 transcripts are continuously synthesized but are targeted for degradation rather than splicing and polyadenylation. Turnover of crs1 RNA requires the exosome, as do previously described nuclear surveillance and silencing mechanisms, but does not involve a noncanonical poly(A) polymerase. Instead, crs1 transcripts are targeted for destruction by a factor previously implicated in turnover of meiotic RNAs in growing cells. Like exosome mutants, mmi1 mutants splice and polyadenylate vegetative crs1 transcripts. Two regulatory elements are located at the 3' end of the crs1 gene, consistent with the increased accumulation of spliced RNA in polyadenylation factor mutants. This highly integrated regulatory strategy may ensure a rapid response to adverse conditions, thereby guaranteeing survival.

Gallic acid, a major component of Toona sinensis leaf extracts, contains a ROS-mediated anti-cancer activity in human prostate cancer cells.

Prostate cancer, the most frequently diagnosed malignancy in elderly males of the United States, has become a major health issue in Asia. Previous studies have demonstrated that leaf extracts of Toona sinensis Roem. contain cytotoxic activity on several cancer cells including prostate cancer cells. In this study, gallic acid is identified as the major anti-cancer compound in T. sinensis leaf extracts. It is cytotoxic to DU145 prostate cancer cells, through generation of reactive oxygen species (ROS) and mitochondria-mediated apoptosis, which were reversed by antioxidants catalase and N-acetylcysteine. Furthermore, gallic acid is shown to block the growth of DU145 cells at G2/M phases by activating Chk1 and Chk2 and inhibiting Cdc25C and Cdc2 activities. In addition, gallic acid has a synergistic effect with doxorubicin in suppressing the growth of DU145 cells. Taken together, our results suggest that gallic acid has the potential to be developed into an anti-prostate cancer drug and is worthy of further studies.