The decrease in Rad51 and DNA ligase IV nuclear protein expression in Msh2 knockdown HC11 cells induced the low CRISPR/Cas9-mediated knock-in efficiency at the ß-casein gene locus.

Objective: Successful gene editing technology is crucial in molecular biology and related fields. An essential part of an efficient knock-in system is increasing homologous recombination (HR) efficiency in the double-strand break (DSB) repair pathways. Interestingly, HR is closely related to the DNA mismatch repair (MMR) pathway, whereby MMR-related gene Msh2 recognizes a mismatch of nucleotides in recombinant intermediates or gene conversion formed during HR. This study aimed to investigate how the knockdown of Msh2 affects HR-mediated knock-in efficiency at the mouse ß-casein locus. Therefore, we investigated the effect of inhibiting Msh2 expression on the expression of the HR-related gene Rad51 and the key enzyme DNA ligase IV involved in non-homologous end joining (NHEJ). Methods: The knock-in vector targeting the mouse ß-casein gene locus, programmed guide RNA, and Msh2 siRNA expression vector were co-transfected in HC11 cells, or only the Msh2 siRNA expression vector was transfected. Knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA and protein expression of Msh2, HR-related gene Rad51, and NHEJ-related gene DNA ligase IV were evaluated by quantitative reverse transcription PCR (RT-qPCR) and Western blot analysis. Results: The knock-in vector efficiency at the mouse ß-casein gene locus significantly decreased upon Msh2 knockdown in HC11 mouse mammary epithelial cells (HC11 cell). Additionally, the knockdown of the DNA MMR-related gene Msh2 protein significantly downregulated the nuclear protein expression of the HR-related Rad51 and NHEJ-related DNA ligase IV genes. Conclusion: The decreased Msh2 protein expression in the nucleus downregulated the Rad51 and ligase IV protein expressions. Consequently, reduced Rad51 expression results in decreased knock-in efficiency.

Development of a prognostic model related to homologous recombination deficiency in glioma based on multiple machine learning.

Background: Despite advances in neuro-oncology, treatments of glioma and tools for predicting the outcome of patients remain limited. The objective of this research is to construct a prognostic model for glioma using the Homologous Recombination Deficiency (HRD) score and validate its predictive capability for glioma. Methods: We consolidated glioma datasets from TCGA, various cancer types for pan-cancer HRD analysis, and two additional glioma RNAseq datasets from GEO and CGGA databases. HRD scores, mutation data, and other genomic indices were calculated. Using machine learning algorithms, we identified signature genes and constructed an HRD-related prognostic risk model. The model's performance was validated across multiple cohorts. We also assessed immune infiltration and conducted molecular docking to identify potential therapeutic agents. Results: Our analysis established a correlation between higher HRD scores and genomic instability in gliomas. The model, based on machine learning algorithms, identified seven key genes, significantly predicting patient prognosis. Moreover, the HRD score prognostic model surpassed other models in terms of prediction efficacy across different cancers. Differential immune cell infiltration patterns were observed between HRD risk groups, with potential implications for immunotherapy. Molecular docking highlighted several compounds, notably Panobinostat, as promising for high-risk patients. Conclusions: The prognostic model based on the HRD score threshold and associated genes in glioma offers new insights into the genomic and immunological landscapes, potentially guiding therapeutic strategies. The differential immune profiles associated with HRD-risk groups could inform immunotherapeutic interventions, with our findings paving the way for personalized medicine in glioma treatment.

NBS1 dePARylation by NUDT16 is critical for DNA double-strand break repair.

NBS1, a protein linked to the autosomal recessive disorder Nijmegen breakage syndrome, plays an essential role in the DNA damage response and DNA repair. Despite its importance, the mechanisms regulating NBS1 and the impact of this regulation on DNA repair processes remain obscure. In this study, we discovered a new post-translational modification of NBS1, ADP-ribosylation. This modification can be removed by the NUDT16 hydrolase. The loss of NUDT16 results in a reduction of NBS1 protein levels due to NBS1 PARylation-dependent ubiquitination and degradation, which is mediated by the PAR-binding E3 ubiquitin ligase, RNF146. Importantly, ADP-ribosylation of NBS1 is crucial for its localization at DSBs and its involvement in homologous recombination (HR) repair. Additionally, the NUDT16-NBS1 interaction is regulated in response to DNA damage, providing further rationale for NBS1 regulation by NUDT16 hydrolase. In summary, our study unveils the critical role of NUDT16 in governing both the stability of NBS1 and recruitment of NBS1 to DNA double-strand breaks, providing novel insights into the regulation of NBS1 in the HR repair pathway.

The complete mitochondrial genome of Castanopsis carlesii and Castanea henryi reveals the rearrangement and size differences of mitochondrial DNA molecules.

BACKGROUND: Castanopsis carlesii is a dominant tree species in subtropical evergreen broad-leaved forests and holds significant ecological value. It serves as an excellent timber tree species and raw material for cultivating edible fungi. Henry Chinquapin (Castanea henryi) wood is known for its hardness and resistance to water and moisture, making it an exceptional timber species. Additionally, its fruit has a sweet and fruity taste, making it a valuable food source. However, the mitogenomes of these species have not been previously reported. To gain a better understanding of them, this study successfully assembled high-quality mitogenomes of C. carlesii and Ca. henryi for the first time. RESULTS: Our research reveals that the mitochondrial DNA (mtDNA) of C. carlesii exhibits a unique multi-branched conformation, while Ca. henryi primarily exists in the form of two independent molecules that can be further divided into three independent molecules through one pair of long repetitive sequences. The size of the mitogenomes of C. carlesii and Ca. henryi are 592,702 bp and 379,929 bp respectively, which are currently the largest and smallest Fagaceae mitogenomes recorded thus far. The primary factor influencing mitogenome size is dispersed repeats. Comparison with published mitogenomes from closely related species highlights differences in size, gene loss patterns, codon usage preferences, repetitive sequences, as well as mitochondrial plastid DNA segments (MTPTs). CONCLUSIONS: Our study enhances the understanding of mitogenome structure and evolution in Fagaceae, laying a crucial foundation for future research on cell respiration, disease resistance, and other traits in this family.

Poly(Adenosine Diphosphate Ribose) Polymerase (PARP) Inhibitors in the Treatment of Advanced Ovarian Cancer: A Narrative Review.

Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors have appeared as a revolutionary approach to treating advanced ovarian cancer, particularly in patients with breast cancer (BRCA) mutations and homologous recombination deficiency (HRD). This narrative review explores PARP inhibitors' clinical efficiency, safety, and changing role in this context. PARP inhibitors, such as olaparib, niraparib, or rucaparib, provide considerable benefits regarding progression-free survival expansion and overall outcomes improvement in first-line maintenance and recurrent settings. The underlying mechanisms, patient selection criteria, and resistance patterns are discussed, alongside insights into combination therapies to overcome resistance and enhance therapeutic efficacy. Ongoing clinical trials and future potential for personalized therapy approaches using PARP inhibitors for advanced ovarian cancer are also highlighted. However, despite these drugs' phenomenal ability to revolutionize treatment protocols for such cancer types, several challenges remain: toxicity management, cost, and development of resistance will require more research to optimize their use or broaden patient populations who can benefit from them.

Improved methods for genetic manipulation of the alkaliphile Halalkalibacterium halodurans.

An improved approach was developed for the genetic manipulation of the Gram-positive extremophile Halalkalibacterium halodurans (formerly called Bacillus halodurans). We describe an allelic replacement method originally developed for Staphylococcus aureus that allows the deletion, mutation, or insertion of genes without leaving markers or other genetic scars. In addition, a protocol for rapid in vitro plasmid methylation and transformation is presented. The combined methods allow the routine genetic manipulation of H. halodurans from initial transformation to the desired strain in 8 days. These methods improve H. halodurans as a model organism for the study of extremophiles.

Whole-genome sequencing resolves biochemical misidentification of Neisseria species from urogenital specimens.

Neisseria meningitidis (Nm) and Neisseria gonorrhoeae (Ng) are human pathogens that sometimes occupy the same anatomical niche. Ng, the causative agent of gonorrhea, infects 87 million individuals annually worldwide and is an urgent threat due to increasing drug resistance. Ng is a pathogen of the urogenital tract and may infect the oropharyngeal or rectal site, often asymptomatically. Conversely, Nm is an opportunistic pathogen. While often a commensal in the oropharyngeal tract, it is also the leading cause of bacterial meningitis with 1.2 million cases globally, causing significant morbidity and mortality. Horizontal gene transfer (HGT) is likely to occur between Ng and Nm due to their shared anatomical niches and genetic similarity, which poses challenges for accurate detection and treatment. Routine surveillance through the Gonococcal Isolate Surveillance Project and Strengthening the U.S. Response to Resistant Gonorrhea detected six concerning urogenital Neisseria isolates with contradicting species identification in Milwaukee (MIL). While all six isolates were positive for Ng using nucleic acid amplification testing (NAAT) and matrix-assisted laser desorption/ionization time of flight identified the isolates as Ng, two biochemical tests, Gonochek-II and API NH, classified them as Nm. To address this discrepancy, we performed whole-genome sequencing (WGS) using Illumina MiSeq on all isolates and employed various bioinformatics tools. Species detection analysis using BMScan, which uses WGS data, identified all isolates as Ng. Furthermore, Kraken revealed over 98% of WGS reads mapped to the Ng genome and <1% to Nm. Recombination analysis identified putative HGT in all MIL isolates within the γ-glutamyl transpeptidase (ggt) gene, a key component in the biochemical tests used to differentiate between Nm and Ng. Further analysis identified Nm as the source of HGT event. Specifically, the active Nm ggt gene replaced the Ng pseudogenes, ggt1 and ggt2. Together, this study demonstrates that closely related Neisseria species sharing a niche underwent HGT, which led to the misidentification of species following biochemical testing. Importantly, NAAT accurately detected Ng. The misidentification highlights the importance of using WGS to continually evaluate diagnostic or bacterial identification tests.

DNA contamination within recombinant adeno-associated virus preparations correlates with decreased CD34+ cell clonogenic potential.

Recombinant adeno-associated viruses (rAAV) are promising for applications in many genome editing techniques through their effectiveness as carriers of DNA homologous donors into primary hematopoietic stem and progenitor cells (HSPCs), but they have many outstanding concerns. Specifically, their biomanufacturing and the variety of factors that influence the quality and consistency of rAAV preps are in question. During the process of rAAV packaging, a cell line is transfected with several DNA plasmids that collectively encode all the necessary information to allow for viral packaging. Ideally, this process results in the packaging of complete viral particles only containing rAAV genomes; however, this is not the case. Through this study, we were able to leverage single-stranded virus (SSV) sequencing, a next-generation sequencing-based method to quantify all DNA species present within rAAV preps. From this, it was determined that much of the DNA within some rAAV preps is not vector-genome derived, and there is wide variability in the contamination by DNA across various preps. Furthermore, we demonstrate that transducing CD34+ HSPCs with preps with higher contaminating DNA resulted in decreased clonogenic potential, altered transcriptomic profiles, and decreased genomic editing. Collectively, this study characterized the effects of DNA contamination within rAAV preps on CD34+ HSPC cellular potential.

Prevalence and outcomes of germline pathogenic variants of homologous recombination repair genes in ovarian cancer.

Germline pathogenic variants (PVs) are pivotal in gynecological oncology. We focused on the prevalence, clinicopathological features, and survival impact of homologous recombination repair (HRR) PVs in patients with epithelial ovarian cancer (EOC). This was a multicenter retrospective cohort study, and 1248 patients with EOC were registered. Eligible patients (n = 1112) underwent germline DNA analysis for 26 cancer predisposition genes, including nine HRR-related genes, such as BRCA1/2, BRIP1, PALB2, RAD51C/D, and ATM. The associations between clinicopathological factors and HRR-related PVs were examined. Kaplan-Meier and Cox regression analyses were conducted. Among 1091 analyzed patients, 153 (14.0%) carried PVs and 140 (12.8%) were HRR-related. HRR-PV-positive status significantly correlated with serous carcinoma (22.9% vs. 4.8%, P < 0.0001) and advanced disease (18.5% vs. 5.9%, P < 0.0001). The HRR-PV-positive group exhibited higher prevalence of personal breast (12.9%) and familial breast/ovarian (29.2%) cancer history. HRR status independently improved overall survival in stage III/IV disease (P = 0.04) but not progression-free survival. HRR-related germline PVs exhibit distinct clinicopathological features with survival implications. Variants were significantly associated with serous carcinoma and advanced disease, underscoring the importance of genetic testing to develop individualized EOC treatment strategies. Considering the study period (2000-2019), the limited use of bevacizumab and poly (ADP-ribose) polymerase inhibitors as maintenance therapy should be recognized.

Interchromosomal segmental duplication drives translocation and loss of P. falciparum histidine-rich protein 3.

Most malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and PfHRP3, but deletions of pfhrp2 and phfrp3 genes make parasites undetectable by RDTs. We analyzed 19,313 public whole-genome-sequenced P. falciparum field samples to understand these deletions better. Pfhrp2 deletion only occurred by chromosomal breakage with subsequent telomere healing. Pfhrp3 deletions involved loss from pfhrp3 to the telomere and showed three patterns: no other associated rearrangement with evidence of telomere healing at breakpoint (Asia; Pattern 13-TARE1); associated with duplication of a chromosome 5 segment containing multidrug-resistant-1 gene (Asia; Pattern 13-5++); and most commonly, associated with duplication of a chromosome 11 segment (Americas/Africa; Pattern 13-11++). We confirmed a 13-11 hybrid chromosome with long-read sequencing, consistent with a translocation product arising from recombination between large interchromosomal ribosome-containing segmental duplications. Within most 13-11++ parasites, the duplicated chromosome 11 segments were identical. Across parasites, multiple distinct haplotype groupings were consistent with emergence due to clonal expansion of progeny from intrastrain meiotic recombination. Together, these observations suggest negative selection normally removes 13-11++pfhrp3 deletions, and specific conditions are needed for their emergence and spread including low transmission, findings that can help refine surveillance strategies.

DOT1L: orchestrating methylation-dependent radiotheRAPy responses via BRCA1.

Breast Cancer Type 1 Susceptibility Protein (BRCA)-1 existing in several functionally distinct complexes, promotes DNA repair of DNA double-strand breaks (DSBs). A recent study by Tang and colleagues identifies the lysine methyltransferase Disruptor of Telomeric Silencing 1-Like (DOT1L) involved in modifying Receptor-Associated Protein 80 (RAP80) to promote BRCA1-A complex localization and repair functions at DNA breaks. This study illuminates a potential therapeutic target for cancer radiotherapy.

The DNA Damage Repair Function of Fission Yeast CK1 Involves Targeting Arp8, a Subunit of the INO80 Chromatin Remodeling Complex.

The CK1 family are conserved serine/threonine kinases with numerous substrates and cellular functions. The fission yeast CK1 orthologues Hhp1 and Hhp2 were first characterized as regulators of DNA repair, but the mechanism(s) by which CK1 activity promotes DNA repair had not been investigated. Here, we found that deleting Hhp1 and Hhp2 or inhibiting CK1 catalytic activities in yeast or in human cells increased double-strand breaks (DSBs). The primary pathways to repair DSBs, homologous recombination and nonhomologous end joining, were both less efficient in cells lacking Hhp1 and Hhp2 activity. To understand how Hhp1 and Hhp2 promote DNA damage repair, we identified new substrates of these enzymes using quantitative phosphoproteomics. We confirmed that Arp8, a component of the INO80 chromatin remodeling complex, is a bona fide substrate of Hhp1 and Hhp2 important for DNA repair. Our data suggest that Hhp1 and Hhp2 facilitate DNA repair by phosphorylating multiple substrates, including Arp8.

Template plasmids optimized for deletion of multiple genes in yeast Saccharomyces cerevisiae.

For Saccharomyces cerevisiae, gene knockout is routinely performed by transformation with a linear DNA cassette consisting of a selection marker gene flanked by upstream and downstream sequences homologous to a target gene. Over the years, several plasmid sets containing a variety of selection marker genes have been developed. Targeting fidelity under this strategy was high when performing the first gene knockout in a strain. However, we found that targeting fidelity decreased substantially when performing subsequent gene knockouts. The majority of the transformants were "incorrect," in which the new selection marker gene replaced a pre-existing selection marker gene instead of its intended target. This was caused by the presence of shared regions in the knockout DNA cassettes. To minimize shared regions among knockout cassettes, we developed a set of template plasmids, in which each selection marker open reading frame is flanked by a unique promoter/terminator combination. Our SJZ series templates cover eight selection markers, namely, URA3 (C. a.), TRP1 (K.l.), his5 (S.p.), LEU2 (K.l.), nat, hph, kan, and amdS. When using our templates, targeting fidelity in subsequent gene knockouts was restored to as high as that of the first knockout, with essentially all the transformants being correct. Our templates can therefore bring efficiency improvements in future research projects involving multi-gene knockouts. IMPORTANCE: When knocking out multiple genes in yeast, recombination among selection markers produces a large portion of false-positive transformants. We developed a new set of templates designed to minimize shared regions among selection markers. The use of this new template set resulted in essentially all transformants being correct knockouts.

The epistatic relationship of Drosophila melanogaster CtIP and Rif1 in homology-directed repair of DNA double-strand breaks.

Double-strand breaks (DSBs) are genotoxic DNA lesions that pose significant threats to genomic stability, necessitating precise and efficient repair mechanisms to prevent cell death or mutations. DSBs are repaired through nonhomologous end-joining (NHEJ) or homology-directed repair (HDR), which includes homologous recombination (HR) and single-strand annealing (SSA). CtIP and Rif1 are conserved proteins implicated in DSB repair pathway choice, possibly through redundant roles in promoting DNA end-resection required for HDR. Although the roles of these proteins have been well-established in other organisms, the role of Rif1 and its potential redundancies with CtIP in Drosophila melanogaster remain elusive. To examine the roles of DmCtIP and DmRif1 in DSB repair, this study employed the direct repeat of white (DR-white) assay, tracking across indels by decomposition (TIDE) analysis, and P{wIw_2 kb 3'} assay to track repair outcomes in HR, NHEJ, and SSA, respectively. These experiments were performed in DmCtIPΔ/Δ single mutants, DmRif1Δ/Δ single mutants, and DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants. This work demonstrates significant defects in both HR and SSA repair in DmCtIPΔ/Δ and DmRif1Δ/Δ single mutants. However, experiments in DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants reveal that DmCtIP is epistatic to DmRif1 in promoting HDR. Overall, this study concludes that DmRif1 and DmCtIP do not perform their activities in a redundant pathway, but rather DmCtIP is the main driver in promoting HR and SSA, most likely through its role in end resection.

Prediction of homologous recombination deficiency from routine histology with attention-based multiple instance learning in nine different tumor types.

BACKGROUND: Homologous recombination deficiency (HRD) is recognized as a pan-cancer predictive biomarker that potentially indicates who could benefit from treatment with PARP inhibitors (PARPi). Despite its clinical significance, HRD testing is highly complex. Here, we investigated in a proof-of-concept study whether Deep Learning (DL) can predict HRD status solely based on routine hematoxylin & eosin (H&E) histology images across nine different cancer types. METHODS: We developed a deep learning pipeline with attention-weighted multiple instance learning (attMIL) to predict HRD status from histology images. As part of our approach, we calculated a genomic scar HRD score by combining loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST) from whole genome sequencing (WGS) data of n = 5209 patients across two independent cohorts. The model's effectiveness was evaluated using the area under the receiver operating characteristic curve (AUROC), focusing on its accuracy in predicting genomic HRD against a clinically recognized cutoff value. RESULTS: Our study demonstrated the predictability of genomic HRD status in endometrial, pancreatic, and lung cancers reaching cross-validated AUROCs of 0.79, 0.58, and 0.66, respectively. These predictions generalized well to an external cohort, with AUROCs of 0.93, 0.81, and 0.73. Moreover, a breast cancer-trained image-based HRD classifier yielded an AUROC of 0.78 in the internal validation cohort and was able to predict HRD in endometrial, prostate, and pancreatic cancer with AUROCs of 0.87, 0.84, and 0.67, indicating that a shared HRD-like phenotype occurs across these tumor entities. CONCLUSIONS: This study establishes that HRD can be directly predicted from H&E slides using attMIL, demonstrating its applicability across nine different tumor types.

Long-read sequencing of an advanced cancer cohort resolves rearrangements, unravels haplotypes, and reveals methylation landscapes.

The Long-Read Personalized OncoGenomics (POG) dataset comprises a cohort of 189 patient tumors and 41 matched normal samples sequenced using the Oxford Nanopore Technologies PromethION platform. This dataset from the POG program and the Marathon of Hope Cancer Centres Network includes DNA and RNA short-read sequence data, analytics, and clinical information. We show the potential of long-read sequencing for resolving complex cancer-related structural variants, viral integrations, and extrachromosomal circular DNA. Long-range phasing facilitates the discovery of allelically differentially methylated regions (aDMRs) and allele-specific expression, including recurrent aDMRs in the cancer genes RET and CDKN2A. Germline promoter methylation in MLH1 can be directly observed in Lynch syndrome. Promoter methylation in BRCA1 and RAD51C is a likely driver behind homologous recombination deficiency where no coding driver mutation was found. This dataset demonstrates applications for long-read sequencing in precision medicine and is available as a resource for developing analytical approaches using this technology.

The association of the chemotherapy response score and homologous recombination deficiency in patients undergoing interval tumor reductive surgery following neoadjuvant chemotherapy.

OBJECTIVES: In patients undergoing interval tumor reductive surgery, a good response to neoadjuvant chemotherapy may limit available tumor for homologous recombination deficiency testing. The objective of this study was to assess whether the chemotherapy response score predicts homologous recombination status. METHODS: We identified patients with advanced epithelial ovarian cancer (diagnosed January 2019 to 20 June 2023) who received neoadjuvant chemotherapy, underwent interval surgery, and for whom a chemotherapy response score was reported (1=no or minimal tumor response, 2=appreciable tumor response, 3=complete or near complete response with no residual tumor). Comparisons were made using ANOVAs or Kruskal-Wallis test for continuous variables and χ2 or Fisher's exact test for categorical variables. RESULTS: The cohort consisted of 234 patients with advanced ovarian cancer who underwent interval surgery following neoadjuvant chemotherapy. Of those who underwent germline genetic testing, 22% (51/232) had a pathogenic BRCA1 or BRCA2 mutation and of those with tumors sent for testing, 65% were found to have homologous recombination deficiency (66/146). With increasing chemotherapy response scores, a higher likelihood of a complete gross resection was observed (50% (chemotherapy response score, CRS 1) vs 77% (CRS 2) vs 88% (CRS 3), p<0.001). On multivariable analysis, CRS 2 (adjusted odds ratio=3.28, 95% CI 1.12 to 9.60, p=0.03) and CRS 3 (5.83, 1.79 to 18.93, p=0.003) were independently associated with homologous recombination deficiency compared with CRS 1. CONCLUSION: A positive response to chemotherapy at the time of interval tumor reductive surgery defined by the chemotherapy response score was associated with homologous recombination status and the likelihood of achieving a complete gross resection.

LncRNA LINC01664 promotes cancer resistance through facilitating homologous recombination-mediated DNA repair.

The intracellular responses to DNA double-strand breaks (DSB) repair are crucial for genomic stability and play an essential role in cancer resistance. In addition to canonical DSB repair proteins, long non-coding RNAs (lncRNAs) have been found to be involved in this sophisticated network. In the present study, we performed a loss-of-function screen for a customized siRNA Premix Library to identify lncRNAs that participate in homologous recombination (HR) process. Among the candidates, we identified LINC01664 as a novel lncRNA required for HR repair. Furthermore, LINC01664 knockdown significantly increased the sensitivity of cancer cells to DNA damage agents such as ionizing radiation and genotoxic drugs. Mechanistically, LINC01664 interacted with Sirt1 promoter and then activated Sirt1 transcription, which contributed to HR-mediated DNA damage repair. In summary, our findings revealed a new mechanism of LINC01664 in DNA damage repair, providing evidence for a potential therapeutic strategy for eliminating the treatment bottlenecks caused by cancer resistance to chemotherapy and radiotherapy.

Emergence and evolution of mosaic penA-60 and penA-237 alleles in a Neisseria gonorrhoeae core genogroup that was historically susceptible to extended spectrum cephalosporins.

Introduction: Neisseria gonorrhoeae (Ng) has successively developed resistance to all previously recommended antimicrobial therapies, with ceftriaxone being the last option for monotherapy of gonorrhea. Global emergence and international spread of the FC428 clone derived mosaic penA-60 allele, associated with highlevel ceftriaxone minimum inhibitory concentrations (MICs) in non FC428 clone Ng lineages, has become an increasing concern. The penA-60 allele carrying Ng was first identified in the U.S. in Las Vegas, Nevada (2019; GCWGS-102723), with a multi-locus sequence type (MLST)-1901 strain, in a non FC428 clone Ng lineage, which is associated with a historically ceftriaxone susceptible core genogroup. Later in 2022, an allele genetically similar to penA-60, mosaic penA-237, was identified in the UK (H22-722) and France (F92) with high-level ceftriaxone MICs and both belonged to MLST-1901. Methods: In this study, we assessed phylogenomic relatedness and antimicrobial resistance (AMR) determinant profiles of these three isolates with high-level ceftriaxone MICs among a global collection of 2,104 genomes belonging to the MLST-1901 core genome cluster group 31, which includes strains separated by a locus threshold of 200 or fewer differences (Ng_cgc_200). Recombination events in and around the penA coding region were catalogued and potential sources of inter species recombinant DNA were also inferred. Results: The global population structure of MLST-1901 core genogroup falls into 4 major lineages. Isolates GCWGS-10723, F92, and H22-722 clustered within Lineage 1, which was dominated by non-mosaic penA-5 alleles. These three isolates formed a clade within Lineage 1 that consisted of isolates from North America and southeast Asia. Neisseria subflava and Neisseria sicca were identified as likely progenitors of two independent recombination events that may have led to the generation of mosaic penA-60 and penA-237, within a possible non-mosaic penA-5 background. Discussions: Our study suggests that there are multiple evolutionary pathways that could generate concerning mosaic penA alleles via homologous recombination of historically susceptible Ng lineages with Neisseria commensals. Enhanced surveillance of gonococcal strains and Neisseria commensals is crucial for understanding of the evolution of AMR, particularly in less-studied regions (e.g., Asia), where high-level ceftriaxone MICs and multi-drug resistance are more prevalent.

CRISPR/Cas9 improves targeted knock-in efficiency in Aspergillus oryzae.

Aspergillus oryzae is an important fungus in food and industrial enzyme production. In A. oryzae, targeted knock-in transformation is primarily limited to homologous recombination (HR)-based systems, in which non-homologous end-joining (NHEJ)-disruptant hosts are required. However, preparation of hosts and transformation templates for such systems is laborious, in addition to other disadvantages. In the present study, we examined alternative targeted knock-in mediated by CRISPR/Cas9, in which a microhomology-mediated end-joining (MMEJ) and single-strand annealing (SSA) repair system was employed. This approach enabled the efficient development of targeted knock-in transformants without host preparation using only a short homology template. We conclude that this new method could be applied to facilitate the transformation of A. oryzae, and will make it easier to acquire targeted knock-in transformants, especially from industrially important non-model strains.