Evaluation of the paired-Cas9 nickase and RNA-guided FokI genome editing tools in precise integration of an anti-CD52 bicistronic monoclonal antibody expression construct at Chinese hamster ovary cells 18S rDNA locus.

INTRODUCTION: The aim of this study was to compare two CRISPR/Cas9-based orthogonal strategies, paired-Cas9 nickase (paired-Cas9n) and RNA-guided FokI (RFN), in targeting 18S rDNA locus in Chinese hamster ovary (CHO) cells and precisely integrating a bicistronic anti-CD52 monoclonal antibody (mAb) expression cassette into this locus. METHODS: T7E1 and high-resolution melt (HRM) assays were used to compare the ability of mentioned systems in inducing double-strand break (DSB) at the target site. Moreover, 5'- and 3'-junction polymerase chain reactions (PCR) were used to verify the accuracy of the targeted integration of the mAb expression cassette into the 18S rDNA locus. Finally, anti-CD52 mAb gene copy number was measured and, its expression was analyzed using ELISA and western blot assays. RESULTS: Our results indicated that both paired-Cas9n and RFN induced DSB at the target site albeit RFN performance was slightly more efficient in HRM analysis. We also confirmed that the anti-CD52 mAb cassette was accurately integrated at the 18S rDNA locus and the mAb was expressed successfully in CHO cells. CONCLUSION: Taken together, our findings elucidated that both paired-Cas9n and RFN genome editing tools are promising in targeting the 18S rDNA locus. Site specific integration of the bicistronic anti-CD52 mAb expression cassette at this locus in the CHO-K1 cells was obtained, using RFN. Moreover, proper expression of the anti-CD52 mAb at the 18S rDNA target site can be achieved using the bicistronic internal ribosome entry site (IRES)-based vector system.

Efficient and safe therapeutic use of paired Cas9-nickases for primary hyperoxaluria type 1.

The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. In addition, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.

Genome-wide DNase I-hypersensitive site assay reveals distinct genomic distributions and functional features of open chromatin in autopolyploid sugarcane.

The characterization of cis-regulatory DNA elements (CREs) is essential for deciphering the regulation of gene expression in eukaryotes. Although there have been endeavors to identify CREs in plants, the properties of CREs in polyploid genomes are still largely unknown. Here, we conducted the genome-wide identification of DNase I-hypersensitive sites (DHSs) in leaf and stem tissues of the auto-octoploid species Saccharum officinarum. We revealed that DHSs showed highly similar distributions in the genomes of these two S. officinarum tissues. Notably, we observed that approximately 74% of DHSs were located in distal intergenic regions, suggesting considerable differences in the abundance of distal CREs between S. officinarum and other plants. Leaf- and stem-dependent transcriptional regulatory networks were also developed by mining the binding motifs of transcription factors (TFs) from tissue-specific DHSs. Four TEOSINTE BRANCHED 1, CYCLOIDEA, and PCF1 (TCP) TFs (TCP2, TCP4, TCP7, and TCP14) and two ethylene-responsive factors (ERFs) (ERF109 and ERF03) showed strong causal connections with short binding distances from each other, pointing to their possible roles in the regulatory networks of leaf and stem development. Through functional validation in transiently transgenic protoplasts, we isolate a set of tissue-specific promoters. Overall, the DHS maps presented here offer a global view of the potential transcriptional regulatory elements in polyploid sugarcane and can be expected to serve as a valuable resource for both transcriptional network elucidation and genome editing in sugarcane breeding.

Increasing deletion sizes and the efficiency of CRISPR/Cas9-mediated mutagenesis by SunTag-mediated TREX1 recruitment.

Previously, it has been shown that mutagenesis frequencies can be improved by directly fusing the human exonuclease TREX2 to Cas9, resulting in a strong increase in the frequency of smaller deletions at the cut site. Here, we demonstrate that, by using the SunTag system for recruitment of TREX2, the mutagenesis efficiency can be doubled in comparison to the direct fusion in Arabidopsis thaliana. Therefore, we also tested the efficiency of the system for targeted deletion formation by recruiting two other 3'-5' exonucleases, namely the human TREX1 and E. coli ExoI. It turns out that SunTag-mediated recruitment of TREX1 not only improved the general mutation induction efficiency slightly in comparison to TREX2, but that, more importantly, the mean size of the induced deletions was also enhanced, mainly via an increase of deletions of 25 bp or more. EcExoI also yielded a higher amount of larger deletions. However, only in the case of TREX1 and TREX2, the effect was predominately SunTag-dependent, indicating efficient target-specific recruitment. Using SunTag-mediated TREX1 recruitment at other genomic sites, we were able to obtain similar deletion patterns. Thus, we were able to develop an attractive novel editing tool that is especially useful for obtaining deletions in the range from 20 to 40 bp around the cut site. Such sizes are often required for the manipulation of cis-regulatory elements. This feature is closing an existing gap as previous approaches, based on single nucleases or paired nickases or nucleases, resulted in either shorter or longer deletions, respectively.

Expanding the list of sequence-agnostic enzymes for chromatin conformation capture assays with S1 nuclease.

This study presents a novel approach for mapping global chromatin interactions using S1 nuclease, a sequence-agnostic enzyme. We develop and outline a protocol that leverages S1 nuclease's ability to effectively introduce breaks into both open and closed chromatin regions, allowing for comprehensive profiling of chromatin properties. Our S1 Hi-C method enables the preparation of high-quality Hi-C libraries, marking a significant advancement over previously established DNase I Hi-C protocols. Moreover, S1 nuclease's capability to fragment chromatin to mono-nucleosomes suggests the potential for mapping the three-dimensional organization of the genome at high resolution. This methodology holds promise for an improved understanding of chromatin state-dependent activities and may facilitate the development of new genomic methods.

Split complementation of base editors to minimize off-target edits.

Base editors (BEs) empower the efficient installation of beneficial or corrective point mutations in crop and human genomes. However, conventional BEs can induce unpredictable guide RNA (gRNA)-independent off-target edits in the genome and transcriptome due to spurious activities of BE-enclosing deaminases, and current improvements mostly rely on deaminase-specific mutagenesis or exogenous regulators. Here we developed a split deaminase for safe editing (SAFE) system applicable to BEs containing distinct cytidine or adenosine deaminases, with no need of external regulators. In SAFE, a BE was properly split at a deaminase domain embedded inside a Cas9 nickase, simultaneously fragmenting and deactivating both the deaminase and the Cas9 nickase. The gRNA-conditioned BE reassembly conferred robust on-target editing in plant, human and yeast cells, while minimizing both gRNA-independent and gRNA-dependent off-target DNA/RNA edits. SAFE also substantially increased product purity by eliminating indels. Altogether, SAFE provides a generalizable solution for BEs to suppress off-target editing and improve on-target performance.

Development of a CRISPR/Cas9D10A Nickase (nCas9)-Mediated Genome Editing Tool in Streptomyces.

Streptomycetes have a strong ability to produce a vast array of bioactive natural products (NPs) widely used in agriculture and veterinary/human medicine. The recently developed CRISPR/Cas9-based genome editing tools have greatly facilitated strain improvement for target NP overproduction as well as novel NP discovery in Streptomyces. However, CRISPR/Cas9 shows high toxicity to the host, limiting its application in many Streptomyces strains with a low DNA transformation efficiency. In this study, we developed a low-toxicity CRISPR/Cas9D10A nickase (nCas9)-based genome editing tool in the model strain Streptomyces coelicolor M145. We showed that in the presence of both targeting sgRNA and Cas proteins, utilization of nCas9 instead of Cas9 significantly reduced the toxicity to the host and greatly enhanced cell survival. Using this tool, we achieved deletion of single genes and gene clusters with efficiencies of 87-100 and 63-87%, and simultaneous deletion of two genes or gene clusters with efficiencies of 47 and 43%, respectively. The editing efficiency of nCas9 is comparable to that of the Cas9-mediated editing tool. Finally, the nCas9-based editing tool was successfully applied for genome editing in the industrial rapamycin-producing strain Streptomyces rapamycinicus, in which CRISPR/Cas9 cannot work well. We achieved the deletion of three tested genes with an efficiency of 27.2-30%. Collectively, the CRISPR/nCas9-based editing tool offers a convenient and efficient genetic modification system for the engineering of streptomycetes, particularly those with low DNA transformation efficiency.

Genome-wide chromatin accessibility landscape and dynamics of transcription factor networks during ovule and fiber development in cotton.

BACKGROUND: The development of cotton fiber is regulated by the orchestrated binding of regulatory proteins to cis-regulatory elements associated with developmental genes. The cis-trans regulatory dynamics occurred throughout the course of cotton fiber development are elusive. Here we generated genome-wide high-resolution DNase I hypersensitive sites (DHSs) maps to understand the regulatory mechanisms of cotton ovule and fiber development. RESULTS: We generated DNase I hypersensitive site (DHS) profiles from cotton ovules at 0 and 3 days post anthesis (DPA) and fibers at 8, 12, 15, and 18 DPA. We obtained a total of 1185 million reads and identified a total of 199,351 DHSs through ~ 30% unique mapping reads. It should be noted that more than half of DNase-seq reads mapped multiple genome locations and were not analyzed in order to achieve a high specificity of peak profile and to avoid bias from repetitive genomic regions. Distinct chromatin accessibilities were observed in the ovules (0 and 3 DPA) compared to the fiber elongation stages (8, 12, 15, and 18 DPA). Besides, the chromatin accessibility during ovules was particularly elevated in genomic regions enriched with transposable elements (TEs) and genes in TE-enriched regions were involved in ovule cell division. We analyzed cis-regulatory modules and revealed the influence of hormones on fiber development from the regulatory divergence of transcription factor (TF) motifs. Finally, we constructed a reliable regulatory network of TFs related to ovule and fiber development based on chromatin accessibility and gene co-expression network. From this network, we discovered a novel TF, WRKY46, which may shape fiber development by regulating the lignin content. CONCLUSIONS: Our results not only reveal the contribution of TEs in fiber development, but also predict and validate the TFs related to fiber development, which will benefit the research of cotton fiber molecular breeding.

Targeting NETs using dual-active DNase1 variants.

Background: Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively. Methods: Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences. Results: We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine. Conclusion: Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states.

Development of miniature base editors using engineered IscB nickase.

As a miniature RNA-guided endonuclease, IscB is presumed to be the ancestor of Cas9 and to share similar functions. IscB is less than half the size of Cas9 and thus more suitable for in vivo delivery. However, the poor editing efficiency of IscB in eukaryotic cells limits its in vivo applications. Here we describe the engineering of OgeuIscB and its corresponding ωRNA to develop an IscB system that is highly efficient in mammalian systems, named enIscB. By fusing enIscB with T5 exonuclease (T5E), we found enIscB-T5E exhibited comparable targeting efficiency to SpG Cas9 while showing reduced chromosome translocation effects in human cells. Furthermore, by fusing cytosine or adenosine deaminase with enIscB nickase, we generated miniature IscB-derived base editors (miBEs), exhibiting robust editing efficiency (up to 92%) to induce DNA base conversions. Overall, our work establishes enIscB-T5E and miBEs as versatile tools for genome editing.

Secreted mammalian DNases protect against systemic bacterial infection by digesting biofilms.

Extracellular DNase DNASE1L3 maintains tolerance to self-DNA in humans and mice, whereas the role of its homolog DNASE1 remains controversial, and the overall function of secreted DNases in immunity is unclear. We report that deletion of murine DNASE1 neither caused autoreactivity in isolation nor exacerbated lupus-like disease in DNASE1L3-deficient mice. However, combined deficiency of DNASE1 and DNASE1L3 rendered mice susceptible to bloodstream infection with Staphylococcus aureus. DNASE1/DNASE1L3 double-deficient mice mounted a normal innate response to S. aureus and did not accumulate neutrophil extracellular traps (NETs). However, their kidneys manifested severe pathology, increased bacterial burden, and biofilm-like bacterial lesions that contained bacterial DNA and excluded neutrophils. Furthermore, systemic administration of recombinant DNASE1 protein during S. aureus infection rescued the mortality of DNase-deficient mice and ameliorated the disease in wild-type mice. Thus, DNASE1 and DNASE1L3 jointly facilitate the control of bacterial infection by digesting extracellular microbial DNA in biofilms, suggesting the original evolutionary function of secreted DNases as antimicrobial agents.

Epistatic effects of Siglec-G and DNase1 or DNase1l3 deficiencies in the development of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease that displays considerable heterogeneity not only in its symptoms, but also in its environmental and genetic causes. Studies in SLE patients have revealed that many genetic variants contribute to disease development. However, often its etiology remains unknown. Existing efforts to determine this etiology have focused on SLE in mouse models revealing not only that mutations in specific genes lead to SLE development, but also that epistatic effects of several gene mutations significantly amplify disease manifestation. Genome-wide association studies for SLE have identified loci involved in the two biological processes of immune complex clearance and lymphocyte signaling. Deficiency in an inhibitory receptor expressed on B lymphocytes, Siglec-G, has been shown to trigger SLE development in aging mice, as have mutations in DNA degrading DNase1 and DNase1l3, that are involved in clearance of DNA-containing immune complexes. Here, we analyze the development of SLE-like symptoms in mice deficient in either Siglecg and DNase1 or Siglecg and DNase1l3 to evaluate potential epistatic effects of these genes. We found that germinal center B cells and follicular helper T cells were increased in aging Siglecg -/- x Dnase1 -/- mice. In contrast, anti-dsDNA antibodies and anti-nuclear antibodies were strongly increased in aging Siglecg-/- x Dnase1l3-/- mice, when compared to single-deficient mice. Histological analysis of the kidneys revealed glomerulonephritis in both Siglecg -/- x Dnase1 -/- and Siglecg-/- x Dnase1l3-/- mice, but with a stronger glomerular damage in the latter. Collectively, these findings underscore the impact of the epistatic effects of Siglecg with DNase1 and Dnase1l3 on disease manifestation and highlight the potential combinatory effects of other gene mutations in SLE.

LangMoDHS: A deep learning language model for predicting DNase I hypersensitive sites in mouse genome.

DNase I hypersensitive sites (DHSs) are a specific genomic region, which is critical to detect or understand cis-regulatory elements. Although there are many methods developed to detect DHSs, there is a big gap in practice. We presented a deep learning-based language model for predicting DHSs, named LangMoDHS. The LangMoDHS mainly comprised the convolutional neural network (CNN), the bi-directional long short-term memory (Bi-LSTM) and the feed-forward attention. The CNN and the Bi-LSTM were stacked in a parallel manner, which was helpful to accumulate multiple-view representations from primary DNA sequences. We conducted 5-fold cross-validations and independent tests over 14 tissues and 4 developmental stages. The empirical experiments showed that the LangMoDHS is competitive with or slightly better than the iDHS-Deep, which is the latest method for predicting DHSs. The empirical experiments also implied substantial contribution of the CNN, Bi-LSTM, and attention to DHSs prediction. We implemented the LangMoDHS as a user-friendly web server which is accessible at http:/www.biolscience.cn/LangMoDHS/. We used indices related to information entropy to explore the sequence motif of DHSs. The analysis provided a certain insight into the DHSs.

Engineered CRISPR prime editors with compact, untethered reverse transcriptases.

The CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use this Split-PE system to rapidly identify and engineer more compact prime editor architectures that also broaden the types of RTs used for prime editing.

iDHS-FFLG: Identifying DNase I Hypersensitive Sites by Feature Fusion and Local-Global Feature Extraction Network.

The DNase I hypersensitive sites (DHSs) are active regions on chromatin that have been found to be highly sensitive to DNase I. These regions contain various cis-regulatory elements, including promoters, enhancers and silencers. Accurate identification of DHSs helps researchers better understand the transcriptional machinery of DNA and deepen the knowledge of functional DNA elements in non-coding sequences. Researchers have developed many methods based on traditional experiments and machine learning to identify DHSs. However, low prediction accuracy and robustness limit their application in genetics research. In this paper, a novel computational approach based on deep learning is proposed by feature fusion and local-global feature extraction network to identify DHSs in mouse, named iDHS-FFLG. First of all, multiple binary features of nucleotides are fused to better express sequence information. Then, a network consisting of the convolutional neural network (CNN), bidirectional long short-term memory (BiLSTM) and self-attention mechanism is designed to extract local features and global contextual associations. In the end, the prediction module is applied to distinguish between DHSs and non-DHSs. The results of several experiments demonstrate the superior performances of iDHS-FFLG compared to the latest methods.

TAPE-seq is a cell-based method for predicting genome-wide off-target effects of prime editor.

Prime editors (PEs) are powerful tools that widen the possibilities for sequence modifications during genome editing. Although methods based on the analysis of Cas9 nuclease or nickase activity have been used to predict genome-wide off-target activities of PEs, no tool that directly uses PEs for this purpose has been reported yet. In this study, we present a cell-based assay, named TAgmentation of Prime Editor sequencing (TAPE-seq), that provides genome-wide off-target candidates for PEs. TAPE-seq analyses are successfully performed using many different versions of PEs. The TAPE-seq predictions are compared with results from two other off-site prediction methods, Cas9 nuclease-based GUIDE-seq and Cas9 nickase-based Digenome-seq (nDigenome-seq). TAPE-seq shows a lower miss rate, and a higher area under the receiver operating characteristic curve compared to the other methods. TAPE-seq also identified valid off-target sites that were missed by the other methods.

A method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome.

Methods to reconstruct the mitochondrial DNA (mtDNA) sequence using short-read sequencing come with an inherent bias due to amplification and mapping. They can fail to determine the phase of variants, to capture multiple deletions and to cover the mitochondrial genome evenly. Here we describe a method to target, multiplex and sequence at high coverage full-length human mitochondrial genomes as native single-molecules, utilizing the RNA-guided DNA endonuclease Cas9. Combining Cas9 induced breaks, that define the mtDNA beginning and end of the sequencing reads, as barcodes, we achieve high demultiplexing specificity and delineation of the full-length of the mtDNA, regardless of the structural variant pattern. The long-read sequencing data is analysed with a pipeline where our custom-developed software, baldur, efficiently detects single nucleotide heteroplasmy to below 1%, physically determines phase and can accurately disentangle complex deletions. Our workflow is a tool for studying mtDNA variation and will accelerate mitochondrial research.

Highly efficient generation of isogenic pluripotent stem cell models using prime editing.

The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.

From muscles to nerves, our body is formed of many kinds of cells which can each respond slightly differently to the same harmful genetic changes. Understanding the exact relationship between mutations and cell-type specific function is essential to better grasp how conditions such as Parkinson's disease or amyotrophic lateral sclerosis progress and can be treated. Stem cells could be an important tool in that effort, as they can be directed to mature into many cell types in the laboratory. Yet it remains difficult to precisely introduce disease-relevant mutations in these cells. To remove this obstacle, Li et al. focused on prime editing, a cutting-edge 'search and replace' approach which can introduce new genetic information into a specific DNA sequence. However, it was unclear whether this technique could be used to efficiently create stem cell models of human diseases. A first set of experiments showed that prime editing is superior to conventional approaches when generating mutated genes in stem cells. Li et al. then further improved the efficiency and precision of the method by tweaking how prime editing components are delivered into the cells. The refined approach could be harnessed to quickly generate large numbers of stem cells carrying mutations associated with Parkinson's disease; crucially, prime editing could then also be used to revert a mutated gene back to its healthy form. The improved prime editing approach developed by Li et al. removes a major hurdle for scientists hoping to use stem cells to study genetic diseases. This could potentially help to unlock progress in how we understand and ultimately treat these conditions.

Significant Association of DNASE1 Variable Number Tandem Repeats and Single Nucleotide Polymorphisms With Gastric Cancer.

Introduction: Defects in the apoptotic process are among the most important events involved in carcinogenesis, and defects in DNASE1, as one of the apoptotic machinery components, plays a role in various types of cancer. Previous studies have indicated significant differences in the DNASE1 polymorphisms in different populations. We hypothesized an association of two polymorphic sites in the exon 8 and the intron 4 of the DNASE1 gene with the risk of gastric cancer. Materials and Methods: The study was carried out on 120 gastric cancer patients and 120 age and sex adjusted controls using PCR and RFLP-PCR. Results: The genotype GG (rs1053874) in exon 8 of DNASE1 (odds ratio [95% confidence interval]) 4.65 [2.10-10.29], p < 0.001) and genotype 2/3 of variable number tandem repeat (VNTR) in the intron 4 (3.75 [1.56-9.01], p = 0.003) are both linked to gastric cancer. Conclusion: We propose that both polymorphic sites have a part to play in gastric cancer, and so may be useful diagnosis markers.

Involvement of BldC in the Formation of Physiologically Mature Sporangium in Actinoplanes missouriensis.

AmBldD is a global transcriptional regulator that represses the transcription of several genes required for sporangium formation in Actinoplanes missouriensis. Here, we characterized one of the AmBldD regulons: AMIS_1980, encoding an ortholog of BldC, which is a transcriptional regulator involved in the morphological development of Streptomyces. We determined the transcriptional start point of the bldC ortholog by high-resolution S1 nuclease mapping and found an AmBldD box in its 5'-untranslated region. Reverse transcription-quantitative PCR analysis revealed that the transcription of bldC is activated during sporangium formation. A bldC null mutant (ΔbldC) strain formed normally shaped sporangia, but they exhibited defective sporangium dehiscence; under a dehiscence-inducing condition, the number of spores released from the sporangia of the ΔbldC strain was 2 orders of magnitude lower than that from the sporangia of the wild-type strain. RNA sequencing analysis indicated that BldC functions as a transcriptional activator of several developmental genes, including tcrA, which encodes a key transcriptional activator that regulates sporangium formation, sporangium dehiscence, and spore dormancy. Using electrophoretic mobility shift assay (EMSA), we showed that a recombinant BldC protein directly binds to upstream regions of at least 18 genes, the transcription of which is downregulated in the ΔbldC strain. Furthermore, using DNase I footprinting and EMSA, we demonstrated that BldC binds to the direct repeat sequences containing an AT-rich motif. Thus, BldC is a global regulator that activates the transcription of several genes, some of which are likely to be required for sporangium dehiscence. IMPORTANCE BldC is a global transcriptional regulator that acts as a "brake" in the morphological differentiation of Streptomyces. BldC-like proteins are widely distributed throughout eubacteria, but their orthologs have not been studied outside streptomycetes. Here, we revealed that the BldC ortholog in Actinoplanes missouriensis is essential for sporangium dehiscence and that its regulon is different from the BldC regulon in Streptomyces venezuelae, suggesting that BldC has evolved to play different roles in morphological differentiation between the two genera of filamentous actinomycetes.