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Hard meat has low market value; hence, we used bittern as a novel meat tenderizer for bovine M. semitendinosus, one of a hard muscle. We investigated the effects of beef immersion in bittern, a basic solution primarily comprising MgCl 2 , on textural properties and water-holding capacity. Muscle samples from M. semitendinosus of Holstein steers were immersed in seven different solutions (RO, NaCl, MgCl 2 , red wine, pH 3, bittern, and pH 8) and heated at 80°C for 5min. The pH of the beef and immersion solutions, water-holding capacity, and maximum load of the meat were measured. Although beef immersed in red wine (pH 3) had a lower pH and water-holding capacity, that immersed in bittern (pH 8.4) had a higher pH and higher water holding capacity. These results indicate that immersion in acidic red wine may harden beef and that immersion in basic bittern may be more effective in maintaining water-holding capacity and softening beef., (© 2024 The Author(s). Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)

Dry-cured beef ham quality is determined by moisture content; however, it is hard to do non-destructive monitoring. We investigated whether the internal moisture content of dry-cured beef ham could be determined from the impedance of the surface, which is removed at the time of eating. Supraspinatus muscle samples from Japanese Shorthorn steers were dried for different periods. The samples were then divided into the parts to be removed (surface) and edible parts. Their respective impedance and moisture content were measured, and the correlation between these two parameters was investigated. A negative correlation was observed between the impedance and moisture content of the surface and the shallow and deep sections of the edible part. There was also a negative correlation between the impedance of the surface and the moisture content of the shallow and deep sections of the edible part. Therefore, the results of this study indicate that during drying, the moisture content in the shallow and deep sections of dry-cured hams can be estimated by measuring the impedance of the surface. This non-destructive method of measuring the moisture content of dry-cured hams can help in regulating the moisture content during drying to produce high-quality dry-cured hams., (© 2024 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)

Background: Perigastric abscess caused by delayed perforation after endoscopic submucosal dissection is a very rare complication. In principle, delayed perforation after endoscopic submucosal dissection is treated surgically. Herein, we report a case of perigastric abscess caused by delayed perforation after gastric endoscopic submucosal dissection that was treated conservatively, without perforation closure, and in which the patient was discharged from hospital in a short period., Case Presentation: A-74-year-old Asian man was diagnosed with having early gastric cancer on follow-up endoscopy and was admitted to our hospital for endoscopic resection. Endoscopic submucosal dissection was performed without intraoperative complications. On postoperative day 2, the patient complained of a slight abdominal pain localized to the epigastric region and a small amount of melena. A computed tomography scan revealed the presence of free air in the peritoneal cavity, and a little fluid collection abutting the dorsal area of the stomach. An endoscopy examination showed a deep ulcer with the accumulation of pus, suggesting a perforation in the post-endoscopic submucosal dissection ulcer. We diagnosed a perigastric abscess, caused by delayed perforation after endoscopic submucosal dissection, and opted for conservative treatment, leaving the perforation site open to allow spontaneous drainage from the abscess into the stomach. A follow-up computed tomography scan revealed an encapsuled and localized perigastric abscess on postoperative day 5, and the disappearance of the free air and the regression of the perigastric abscess on postoperative day 7. A follow-up endoscopy examination on postoperative day 7 showed the closure of the perforation. Finally, surgery was avoided, and the patient was discharged on postoperative day 14, after a relatively short hospital stay., Conclusion: Regarding the treatment of perigastric abscess, caused by delayed perforation after endoscopic submucosal dissection, leaving the perforation site open to allow spontaneous drainage may shorten the conservative treatment period., (© 2023. The Author(s).)

Background and study aims  Endoscopic submucosal dissection (ESD) of pharyngeal cancers with conventional endoscopes often is difficult, not only because of the narrow working space, but also because endoscope maneuverability in the pharynx is poor due to interference from the endotracheal tube and/or hyoid bone. However, we hypothesized that those problems could possibly be resolved by use of an ultrathin endoscope for ESD of superficial pharyngeal cancer. The aim of this prospective interventional study was to investigate the feasibility of ESD for superficial pharyngeal cancer using an ultrathin endoscope. Patients and methods  This feasibility study was conducted at NTT Medical Center Tokyo between June 2020 and September 2021, and data from a total of 20 consecutively superficial pharyngeal cancers were analyzed. The primary outcome measure was the R0 resection rate. The ESD completion rate, en bloc resection rate, procedure time, and frequency of intraoperative and postoperative adverse events (AEs) were also evaluated as secondary outcome measures. Results  Data from 16 patients with 20 lesions were included in the analysis. All of the lesions were successfully resected by ultrathin endoscope ESD, and the en bloc and R0 resection rates were 100 % and 85.0 % (17/20), respectively; the procedure time was 37.8 ± 28.2 minutes. No intraoperative or postoperative AEs were encountered in any cases. Conclusions  ESD using an ultrathin endoscope is feasible for superficial pharyngeal cancers and has potential to be a safe and effective treatment option for these cancers., Competing Interests: Competing interests The authors declare that they have no conflict of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Meat pH affects its color, texture, and water-holding capacity; however, a method for nondestructively distinguishing high-pH from normal-pH meat is unknown. We investigated the method of preparing an extensive pH-model of beef immersed in different pH buffers and the relationship between pH of the pH-model beef and impedance, measured using touch-type electrodes. The results showed that a model beef of pH 4.91-6.39 could be obtained when a beef steak (pH 5.68) was immersed in a solution of pH 4.0-7.0. In addition, we found a significant negative correlation between immersion pH and drip loss, and between immersion pH and impedance measured at 50 kHz (P < 0.01). These results provide an estimation equation (y = -16.5x + 195.3) that can be used to nondestructively determine the pH (x) of pH-model beef by measuring its impedance (y) using touch-type electrodes at 50 kHz (P < 0.01). Hence, the study shows the possibility of nondestructively estimating the pH of pH model beef by measuring impedance using touch-type electrodes., (© 2023 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)

Background: Gastric mixed neuroendocrine-non-neuroendocrine neoplasms are rare malignant tumors. The lack of specific findings makes it difficult to diagnose endoscopically. We report the case of early gastric mixed neuroendocrine-non-neuroendocrine neoplasms treated by endoscopic submucosal dissection., Case Presentation: An 81-year-old Japanese female underwent esophagogastroduodenoscopy for screening and was treated with endoscopic submucosal dissection for the diagnosis of early gastric cancer. Histopathologically, the lesion was diagnosed as mixed neuroendocrine-non-neuroendocrine neoplasms (tubular adenocarcinoma 2 60%, endocrine cell carcinoma 40%), pT1b(submucosa (SM) 900 μm), pUL(-), Ly(+), v(-), pHM0, pVM0. After additional surgical resection without adjuvant chemotherapy, she has had no recurrences or metastases for 3 years., Conclusions: Comparing narrow-band imaging magnified endoscopic findings with pathological findings, the depressed area with a lack of surface structure was consistent with the neuroendocrine cell carcinoma component, while narrow-band imaging magnification findings showed non-network vessels. In this case, we examined endoscopic findings of early stage mixed neuroendocrine-non-neuroendocrine neoplasms in detail and compared it with the pathological findings. We believe that these endoscopic findings contribute to the diagnosis of mixed neuroendocrine-non-neuroendocrine neoplasms and can lead to its early detection., (© 2022. The Author(s).)

Genome-wide screening is powerful method used to identify genes and pathways associated with a phenotype of interest. The simple eukaryote Dictyostelium discoideum has a unique life cycle and is often used as a crucial research model for a wide range of biological processes and rare metabolites. To address the inadequacies of conventional genetic screening approaches, we developed a highly efficient CRISPR/Cas9-based genome-wide screening system for Dictyostelium. A genome-wide library of 27,405 gRNAs and a kinase library of 4,582 gRNAs were compiled and mutant pools were generated. The resulting mutants were screened for defects in cell growth and more than 10 candidate genes were identified. Six of these were validated and five recreated mutants presented with growth abnormalities. Finally, the genes implicated in developmental defects were screened to identify the unknown genes associated with a phenotype of interest. These findings demonstrate the potential of the CRISPR/Cas9 system as an efficient genome-wide screening method., (© 2022. The Author(s).)

Fluidic chips have attracted considerable interest in recent years for their potential applications in analytical devices. Previously, we developed a method to fabricate polydimethylsiloxane (PDMS) fluidic chips via templates made using a low-priced commercial Fused Deposition Modeling (FDM) type 3D printer and polymer coatings. However, in general, methods using a template cannot form a flow channel thinner than the template thickness and the width. In this study, the inner wall of a PDMS fluidic chip was coated with PDMS to create a chip with a channel inner diameter smaller than a template. Then, by measuring the flow signal of methyl orange with a single line, the basic properties of the non-coated and coated chip were investigated. As a result, almost the same flow profile was obtained in non-coated and coated chips at the same linear velocity and the same sample injection length. By coating and narrowing the channel width, it is possible to save the amount of sample and carrier solution. Measuring hydrazine in water using a coated chip was also tried. The calibration curve indicated good linearity in the range of 1-6 ppm. However, a concentration point of 7 ppm deviated. The reason for this deviation was presumably due to inadequate mixing of the sample and reagent. By decreasing the flow rate, the calibration curve indicated good linearity in the range of 1-7 ppm., (© 2022. The Author(s).)

Aim: Endoscopic submucosal dissection (ESD) is internationally accepted as a minimally invasive procedure to treat early gastrointestinal cancers endoscopically. Uptake of this procedure in the West is limited. No published data are available in New Zealand. We aimed to evaluate outcomes of this procedure at North Shore Hospital, Auckland., Methods: Following an overseas fellowship training period, we prospectively collected clinical outcomes, complications and defined quality indicators for patients undergoing ESD referred following a multidisciplinary meeting., Results: Between January 2020 until July 2021, 29 ESD procedures were performed in 27 patients, including 14 gastric, five oesophageal and 10 colorectal cases. The mean age was 72 (standard deviation (SD) 10.6). The majority of cases (62%) were done under general anaesthesia. The median lesion size resected was 30mm (interquartile range (IQR) 20-58mm). The pre-endoscopic diagnosis was accurate as confirmed on final histology in 93% of cases. Thirty-four percent of lesions were T1 adenocarcinoma and completely resected. The median total duration of the procedure was 90 minutes (IQR 55-180). 86% of lesions were resected en-bloc. R0 resection was achieved in 72% of cases. All cases with R0 resection were curative except one. Muscular defects without perforation were seen and clipped at the time of endoscopy in 34% of cases. Two perforations were identified and sealed at the time of endoscopy. There were no cases of delayed bleeding, perforation or mortality., Conclusion: These data demonstrate clinical success, efficacy and safety of ESD at our centre. A larger study, comparison with other centres and longer clinical follow-up is required to confirm findings and further improve outcomes., Competing Interests: Nil.

Background & Aims: Cold snare polypectomy (CSP) has become the standard resection method for small colorectal polyps (<10 mm). Sessile serrated lesions (SSL) have low prevalence of advanced histology irrespective of size, and thus could be amenable to CSP. In this study, we evaluated the safety and efficacy of CSP for SSLs ≥10 mm., Methods: Between November 2018 and January 2020, we prospectively enrolled 300 consecutive patients who underwent CSP for 474 SSLs ≥10 mm. To delineate SSL borders, indigo carmine chromoendoscopy and/or image-enhanced endoscopy was conducted. Piecemeal CSP (pCSP) was performed in cases where en-bloc resection was difficult. Biopsy specimens were obtained from the margins of the post-polypectomy defect to confirm complete resection. Surveillance colonoscopy was performed to screen for local recurrence., Results: All lesions were successfully resected using CSP without submucosal injection. The median diameter of the resected lesions was 14 mm, and pCSP was used to resect 106 (22%) lesions. Post-polypectomy biopsies revealed residual serrated tissue in only one case (0.2%). Adverse events included immediate bleeding in 8 (3%) patients; no delayed bleeding events occurred, irrespective of the use of antithrombotic drugs. During a 7-month median follow-up period, surveillance colonoscopies were performed for 384 lesions (81%), and no local recurrences were detected., Conclusions: CSP without submucosal injection is a safe and effective treatment for SSLs ≥10 mm. UMIN Clinical Trials, Number: UMIN000034763., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Although serine ADP-ribosylation (Ser-ADPr) by Poly(ADP-ribose)-polymerases is a cornerstone of the DNA damage response, how this regulates DNA repair and genome stability is unknown. Here, we exploit the ability to manipulate histone genes in Dictyostelium to identify that ADPr of the histone variant H3b at S10 and S28 maintains genome stability by integrating double strand break (DSB) repair with mitotic entry. Given the critical requirement for mitotic H3S10/28 phosphorylation, we develop separation of function mutations that maintain S10 phosphorylation whilst disrupting ADPr. Mechanistically, this reveals a requirement for H3bS10/28 ADPr in non-homologous end-joining by recruiting Ku to DSBs. Moreover, this also identifies H3bS10/S28 ADPr is critical to prevent premature mitotic entry with unresolved DNA damage, thus maintaining genome stability. Together, these data demonstrate how serine ADPr of histones coordinates DNA repair with cell cycle progression to maintain genome stability., (© 2022. The Author(s).)

We investigated the effects of juices of sarunashi (Actinidia arguta) at varying ripening stages (unripe, force-ripe, ripe, and over-ripe) on textural properties and water holding capacity after application to the supraspinatus muscle meat from Japanese Shorthorn steers (n = 6). Following drip and cooking loss measurements, we analyzed the maximum load, gumminess load, cohesiveness, and adhesiveness of the muscle samples. Drip loss did not differ significantly among the groups; however, cooking loss in the over-ripe group was higher than that in the other groups. The maximum load and gumminess load in the over-ripe group were significantly lower than those in the unripe group, but no significant difference was observed for the others. There were no differences in cohesiveness and adhesiveness among the groups. Over-ripened sarunashi juices were found to be more effective for beef tenderization than unripened fruit juice., (© 2022 Japanese Society of Animal Science.)

The color of venison observed at a high-pH is not preferred by consumers. However, the physicochemical properties of venison soaked in a low-pH solution to lower its high-pH are unknown. We investigated the effect of immersion in a low-pH solution on the physicochemical properties of a high-pH venison. The pH of M. longissimus thoracis from seven female wild deer (Cervus nippon) was measured and divided into normal-pH venison and high-pH venison groups. The a* and b* values for high-pH venison were lower than those for normal-pH venison before storage. There was no significant difference in the pH between high-pH venison immersed in a pH 4 solution and normal-pH venison stored. The drip loss of immersed high-pH venison was significantly lower than that of normal-pH venison and high-pH venison. Cooking loss and total loss were the lowest for normal-pH venison and the highest for immersed high-pH venison. The maximum load between normal-pH venison, high-pH venison, and immersed high-pH venison did not differ significantly. Our work showed that meat color became favorable by immersing high-pH venison in a solution of pH 4, followed by heating., (© 2022 Japanese Society of Animal Science.)

Video 1Successful endoscopic submucosal dissection for a tumor completely located within a colonic diverticulum after inversion of the diverticulum using a traction device. After reconfirming that the diverticulum could be inverted into the lumen with forceps, the first endoclip grasping a ring-thread made of dental floss was fixed on the mucosa in the diverticulum. Immediately afterward, a second endoclip was used to grasp the ring-thread and fix it to the opposite colonic wall. By insufflating with air, the lesion within the inverted diverticulum was pulled out of the diverticulum into the colonic lumen, establishing a good field of view for dissection. After local injection of hyaluronic acid, a circumferential incision and dissection were performed using a DualKnife, and the lesion was promptly removed en bloc. The wound was completely closed with endoclips, along with the diverticulum. The total procedure time was 10 minutes, and there were no adverse events., (© 2021 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc.)

Self-aid and mutual assistance among victims are critical for resolving difficulties in the immediate aftermath of a disaster, but individual facilitative factors for such resolution processes are poorly understood. To identify such individual factors in the background (i.e., disaster damage and demographic) and personality domains considering different types of difficulty and resolution, we analyzed survey data collected in the 3-year aftermath of the 2011 Great East Japan Earthquake and Tsunami. We first identified major types of difficulty using a cluster analysis of 18 difficulty domains and then explored individual factors that facilitated six types of resolution (self-help, request for help, help from family, help from an acquaintance, help through cooperation, and public assistance) of these difficulty types. We identified general life difficulties and medico-psychological difficulties as two broad types of difficulty; disaster damage contributed to both types, while some personality factors (e.g., neuroticism) exacerbated the latter. Disaster damage hampered self-resolution and forced a reliance on resolution through cooperation or public assistance. On the other hand, some demographic factors, such as being young and living in a three-generation household, facilitated resolution thorough the family. Several personality factors facilitated different types of resolution, primarily of general life difficulties; the problem-solving factor facilitated self-resolution, altruism, or stubbornness resolutions through requests, leadership resolution through acquaintance, and emotion-regulation resolution through public assistance. Our findings are the first to demonstrate the involvement of different individual, particularly personality, factors in survival in the complex social dynamics of this disaster stage. They may contribute to disaster risk mitigation, allowing sophisticated risk evaluation and community resilience building., Competing Interests: The authors have declared that no competing interests exist.

The development of new techniques to create gene knockouts and knock-ins is essential for successful investigation of gene functions and elucidation of the causes of diseases and their associated fundamental cellular processes. In the biomedical model organism Dictyostelium discoideum , the methodology for gene targeting with homologous recombination to generate mutants is well-established. Recently, we have applied CRISPR/Cas9-mediated approaches in Dictyostelium , allowing the rapid generation of mutants by transiently expressing sgRNA and Cas9 using an all-in-one vector. CRISPR/Cas9 techniques not only provide an alternative to homologous recombination-based gene knockouts but also enable the creation of mutants that were technically unfeasible previously. Herein, we provide a detailed protocol for the CRISPR/Cas9-based method in Dictyostelium . We also describe new tools, including double knockouts using a single CRISPR vector, drug-inducible knockouts, and gene knockdown using CRISPR interference (CRISPRi). We demonstrate the use of these tools for some candidate genes. Our data indicate that more suitable mutants can be rapidly generated using CRISPR/Cas9-based techniques to study gene function in Dictyostelium ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yamashita, Iriki, Kamimura and Muramoto.)

The powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

Video 1Endoscopic transcecal appendectomy under laparoscopic single-port assistance., (© 2021 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc.)

Background and Aim: With the increasing prevalence of persons without Helicobacter pylori (HP) infection, cases of HP-negative gastric cancer are increasing. Although rare, cases of differentiated adenocarcinoma of the antrum have been reported in HP-negative patients. We collected cases with such lesions and investigated their endoscopic and histological features., Methods: Of 1965 consecutive patients with early gastric cancer who underwent endoscopic resection between January 2009 and December 2017, we extracted 9 cases of HP-negative differentiated adenocarcinoma located in the antrum (HPN-DAA). The clinical data, endoscopic findings, and histopathological findings were reviewed., Results: Of the nine patients with HPN-DAA, seven were male, and the median age was 53.8 years. The tumor arose from the pyloric gland mucosa in all cases. According to the endoscopic findings, the lesions were flat-elevated or depressed, mimicking varioliform gastritis. Magnifying endoscopy with narrow-band imaging showed the absence of a clear demarcation line or an irregular microvessel/surface pattern. As for the histopathological findings, eight of the nine lesions were diagnosed as high-grade dysplasia/intraepithelial neoplasia, while the remaining case was diagnosed as tubular adenocarcinoma with submucosal infiltration. The findings of immunohistochemistry confirmed that three cases were of the intestinal mucin phenotype and six were of the mixed gastric and intestinal mucin phenotype., Conclusion: HPN-DAA is a very rarely occurring cancer that had never been recognized earlier. They belong to the new category of HP-negative cancers, and there seems to be a certain number of such cases., (© 2021 The Authors. JGH Open published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Background and study aims  Endoscopic submucosal dissection (ESD) has become the standard treatment for colorectal ESD, but large colorectal tumors remain difficult to remove. We developed a new method, called the palisade technique, by modifying the multiple tunneling technique. In this method, a palisade of submucosal tissue is left beneath the tumor to anchor a dissected specimen, maintaining effective submucosal traction. Patients and methods  The study included 11 patients with large colorectal tumors that were over half the circumference of the colorectal lumen which were treated using the palisade technique from August 2017 to October 2019. Overall resection outcomes were assessed. Results  All 11 lesions were removed en bloc. The R0 resection rate was 45.6 % because of marginal burning of the specimen, but no local recurrence was found after a median observation period of 31 months. The median submucosal dissection time (SDT) and submucosal dissection speed (SDS) were 170 minutes and 23.1 mm 2 /min, respectively. One case of post-ESD hemorrhage was successfully managed endoscopically, and two cases of post-colorectal ESD coagulation syndrome were managed conservatively. Conclusion  The palisade technique can be an effective and safe technique for treating large colorectal tumors that extend over half the luminal circumference., Competing Interests: Competing interests The authors declare that they have no conflict of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commecial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Colorimetric pH strips (MColorpHast™) are very convenient to use, but the decision of pH is based on an individual's color perception and is, therefore, subjective. We developed a pH calculation program for the image of coloring strips on CIE1976 (L*a*b* color space), which slightly underestimated human judgment as the color of pH darker. This image analysis and three individuals' judgments were used for evaluating the strip's features for various qualities of meat from wild animals, and the results were compared with the assessments based on potentiometric pH. In both methods, dipping the strips in distilled water just before use improved the regression coefficient compared with that mentioned in the manual. The image analysis showed higher correlation than human judgments but slightly underestimate pH by a maximum of 0.13 unit from the regression line of the potentiometric pH. In addition, the image analysis revealed meat pigment changed pH higher on the color scale in the lower meat pH region. The strips must be used according to the manual, but dipping is effective when the meat surface is dry, and keeping the strips from touching the meat drip is important in lower pH region because the pigment affects the color of pH., (© 2021 Japanese Society of Animal Science.)

The CRISPR/Cas9 system enables targeted genome modifications across a range of eukaryotes. Although we have reported that transient introduction of all-in-one vectors that express both Cas9 and sgRNAs can efficiently induce multiple gene knockouts in Dictyostelium discoideum, concerns remain about off-target effects and false-positive amplification during mutation detection via PCR. To minimise these effects, we modified the system to permit gene deletions of greater than 1 kb via use of paired sgRNAs and Cas9 nickase. An all-in-one vector expressing the Cas9 nickase and sgRNAs was transiently introduced into D. discoideum, and the resulting mutants showed long deletions with a relatively high efficiency of 10-30%. By further improving the vector, a new dual sgRNA expression vector was also constructed to allow simultaneous insertion of two sgRNAs via one-step cloning. By applying this system, precise point mutations and genomic deletions were generated in the target locus via simultaneous introduction of the vector and a single-stranded oligonucleotide template without integrating a drug resistance cassette. These systems enable simple and straightforward genome editing that requires high specificity, and they can serve as an alternative to the conventional homologous recombination-based gene disruption method in D. discoideum., Competing Interests: The authors have declared that no competing interests exist.