Catalytically inactive Cas9 (dCas9) has become an increasingly popular tool for targeted gene activation/inactivation, live-cell imaging, and base editing. While dCas9 was reported to induce base substitutions and indels, it has not been associated with structural variations. Here, we show that dCas9 impedes replication fork progression to destabilize tandem repeats in budding yeast. When targeted to the CUP1 array comprising ∼16 repeat units, dCas9 induced its contraction in most cells, especially in the presence of nicotinamide. Replication intermediate analysis demonstrated replication fork stalling in the vicinity of dCas9-bound sites. Genetic analysis indicated that while destabilization is counteracted by the replisome progression complex components Ctf4 and Mrc1 and the accessory helicase Rrm3, it involves single-strand annealing by the recombination proteins Rad52 and Rad59. Although dCas9-mediated replication fork stalling is a potential risk in conventional applications, it may serve as a novel tool for both mechanistic studies and manipulation of genomic instability.

Rationale:Metastatic littoral cell angioma (LCA) is extremely rare. No standard therapeutic strategy has been established, and the impact of chemotherapy has not yet been evaluated.Patient concerns:A 61-year-old woman was admitted because of bicytopenia. She had a splenectomy for LCA of the spleen 10 years earlier. Bone marrow aspiration was normal, and a computed tomography (CT) scan showed hepatomegaly with multiple liver tumors.Diagnoses:Liver biopsy samples showed macrophage-like cell infiltration in the hepatic sinusoids. Metastatic LCA was diagnosed based on immunohistochemistry, imaging tests, and the clinical course.Interventions:Immunosuppressive agents, such as prednisolone and cyclosporine, were ineffective. Next, cytotoxic agents, such as etoposide, paclitaxel, and vincristine, were administered.Outcomes:Cytotoxic agents showed a prominent effect against LCA. CT showed improvement of the hepatomegaly, and fluoro-deoxyglucose (FDG) uptake decreased markedly at a follow-up FDG- positron emission tomography (PET) scan.Lessons:Chemotherapeutic treatment based on hemophagocytic syndrome or angiosarcoma might have anti-tumor activity against metastatic LCA. Analysis of the molecular characteristics of this tumor is needed to develop better treatment options.

Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Since each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing. Their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a program to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in the insertion of gene fragments to essential genes (median 52.1%), the complete deletion of an open reading frame (median 78.9%), and the introduction of single amino acid substitutions (median 79.2%). The backbone vectors with the program would provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.

Objectives To evaluate the long-term impact of immunosuppressive therapeutic agents on antibody response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) mRNA vaccination in patients with autoimmune rheumatic diseases (AIRD) in order to propose a strategy for annual vaccination. Methods This prospective multicentre cohort study evaluated the humoral response to second and third BNT162b2 and/or mRNA-1273 vaccines in 382 Japanese AIRD patients classified into 12 different medication groups and in 326 healthy controls (HCs). The third vaccination was administered six months after the second vaccination. Antibody titres were measured using the Elecsys Anti-SARS-CoV-2 S assay. Results The seroconversion rate and antibody titres were lower in AIRD patients than in HCs 3–6 weeks after the second vaccination and 3–6 weeks after the third vaccination. Seroconversion rates were <90% after the third vaccination in patients receiving mycophenolate mofetil and rituximab. A multivariate analysis was conducted, adjusting for age, sex, and glucocorticoid dosage. Antibody levels after the third vaccination were significantly lower in the groups prescribed tumour necrosis factor (TNF) inhibitor with or without methotrexate, abatacept and rituximab or cyclophosphamide than those of HCs. The third vaccination induced an adequate humoral response in patients treated with sulfasalazine, bucillamine, methotrexate monotherapy, iguratimod, interleukin-6 inhibitors or calcineurin inhibitors including tacrolimus. Conclusions Repeated vaccinations in many immunosuppressed patients produced antibody responses similar to those observed in HCs. In contrast, annual vaccination in patients receiving TNF inhibitors, abatacept, mycophenolate mofetil and rituximab may require caution.

Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Since each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing and their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a software to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in both insertion of gene fragments to essential genes and complete deletion of an open reading frame. The backbone vectors with the software would thus provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.