Precise Base Substitution Using CRISPR/Cas-Mediated Base Editor in Rice.

Base editors, CRISPR/Cas-based precise genome editing tools, enable base conversion at a target site without inducing DNA double-strand breaks. The genome editing targetable range is restricted by the requirement for protospacer adjacent motif (PAM) sequence. Cas9 derived from Streptococcus pyogenes (SpCas9)-most widely used for genome editing in many organisms-requires an NGG sequence adjacent to the target site as a PAM. Then, engineered and natural Cas variants with altered PAM recognition are used for base editor to expand the flexibility of base substitution position. In this chapter, we describe a protocol for base editing based on SpCas9-NG, which is a rationally engineered SpCas9 variant that can recognize relaxed NG PAMs.

Peer review bullying threatens diversity, equity, and inclusion.

Bullying during the peer review process is an overlooked form of academic bullying. Measures to limit its negative impact are insufficient, necessitating new initiatives to protect individuals and the integrity of science. If unaddressed, peer review bullying will undermine diversity, equity, and inclusion, particularly harming early-career researchers and minorities.

Exploiting protein domain modularity to enable synthetic control of engineered cells.

The ability to precisely control cellular function in response to external stimuli can enhance the function and safety of cell therapies. In this review, we will detail how the modularity of protein domains has been exploited for cellular control applications, specifically through design of multifunctional synthetic constructs and controllable split moieties. These advances, which build on techniques developed by biologists, protein chemists and drug developers, harness natural evolutionary tendencies of protein domain fusion and fission. In this light, we will highlight recent advances towards the development of novel immunoreceptors, base editors, and cytokines that have achieved intriguing therapeutic potential by taking advantage of well-known protein evolutionary phenomena and have helped cells learn new tricks via synthetic biology. In general, protein modularity, i.e., the relatively facile separation or (re)assembly of functional single protein domains or subdomains, is becoming an enabling phenomenon for cellular engineering by allowing enhanced control of phenotypic responses.

Authorship and Citizen Science: Seven Heuristic Rules.

Citizen science (CS) is an umbrella term for research with a significant amount of contributions from volunteers. Those volunteers can occupy a hybrid role, being both 'researcher' and 'subject' at the same time. This has repercussions for questions about responsibility and credit, e.g. pertaining to the issue of authorship. In this paper, we first review some existing guidelines for authorship and their applicability to CS. Second, we assess the claim that the guidelines from the International Committee of Medical Journal Editors (ICMJE), known as 'the Vancouver guidelines', may lead to exclusion of deserving citizen scientists as authors. We maintain that the idea of including citizen scientists as authors is supported by at least two arguments: transparency and fairness. Third, we argue that it might be plausible to include groups as authors in CS. Fourth and finally, we offer a heuristic list of seven recommendations to be considered when deciding about whom to include as an author of a CS publication.

Streamlined process for effective and strand-selective mitochondrial base editing using mitoBEs.

Mitochondrial base editing tools hold great promise for the investigation and treatment of mitochondrial diseases. Mitochondrial DNA base editors (mitoBEs) integrate a programmable transcription-activator-like effector (TALE) protein with single-stranded DNA deaminase (TadA8e-V106W, APOBEC1, etc.) and nickase (MutH, Nt.BspD6I(C), etc.) to achieve heightened precision and efficiency in mitochondrial base editing. This innovative mitochondrial base editing tool exhibits a number of advantages, including strand-selectivity for editing, high efficiency, and the capacity to perform diverse types of base editing on the mitochondrial genome by employing various deaminases. In this context, we provide a detailed experimental protocol for mitoBEs to assist others in achieving proficient mitochondrial base editing.

Impact of Chromatin Organization and Epigenetics on CRISPR-Cas and TALEN Genome Editing.

DNA lies at the heart of the central dogma of life. Altering DNA can modify the flow of information in fundamental cellular processes such as transcription and translation. The ability to precisely manipulate DNA has led to remarkable advances in treating incurable human genetic ailments and has changed the landscape of biological research. Genome editors such as CRISPR-Cas nucleases and TALENs have become ubiquitous tools in basic and applied biological research and have been translated to the clinic to treat patients. The specificity and modularity of these genome editors have made it possible to efficiently engineer genomic DNA; however, underlying principles governing editing outcomes in eukaryotes are still being uncovered. Editing efficiency can vary from cell type to cell type for the same DNA target sequence, necessitating de novo design and validation efforts. Chromatin structure and epigenetic modifications have been shown to affect the activity of genome editors because of the role they play in hierarchical organization of the underlying DNA. Understanding the nuclear search mechanism of genome editors and their molecular interactions with higher order chromatin will lead to improved models for predicting precise genome editing outcomes. Insights from such studies will unlock the entire genome to be engineered for the creation of novel therapies to treat critical illnesses.

A Primer: Peer Review Process for Advances in Physiology Education.

The dissemination of discipline-focused educational scholarship advances theory and stimulates pedagogical application. The aim of Advances in Physiology Education is to publish manuscripts that advance knowledge and inform educators in the field. This primer is tailored for individuals new to manuscript reviewing, early in their careers, or experienced in reviewing research but not educational manuscripts. Peer reviewing for basic and applied science is akin to evaluating research questions and rigor in teaching and learning studies, with differences in approach and analysis similar to those between biophysics and molecular physiology or cell and integrated physiology. Our purpose is to provide an overview of the review process and expectations. The submission and peer review process involves several steps: authors submit a manuscript, the Editor assigns an Associate Editor, who then assigns peer Reviewers. Reviewers are contacted via email and can accept or decline the invitation. Reviewers evaluate the work's strengths and weaknesses, then independently submit comments and recommendations to the Associate Editor. After review, the Associate Editor collects and weighs Reviewers' comments, sometimes garners additional reviews and input, to make a recommendation to the Editor. The Editor reviews the process, comments, and recommendations to render a final decision. Both authors and Reviewers receive an email with the decision. The editorial staff assist with communication and help track the overall process. Peer review is integral to scientific publishing, ensuring quality and rigor, and reviewing is both a privilege and a responsibility of all in the scientific community.

On RNA-programmable gene modulation as a versatile set of principles targeting muscular dystrophies.

The repurposing of RNA-programmable CRISPR systems from genome editing into epigenome editing tools is gaining pace, including in research and development efforts directed at tackling human disorders. This momentum stems from the increasing knowledge regarding the epigenetic factors and networks underlying cell physiology and disease etiology and from the growing realization that genome editing principles involving chromosomal breaks generated by programmable nucleases are prone to unpredictable genetic changes and outcomes. Hence, engineered CRISPR systems are serving as versatile DNA-targeting scaffolds for heterologous and synthetic effector domains that, via locally recruiting transcription factors and chromatin remodeling complexes, seek interfering with loss-of-function and gain-of-function processes underlying recessive and dominant disorders, respectively. Here, after providing an overview about epigenetic drugs and CRISPR-Cas-based activation and interference platforms, we cover the testing of these platforms in the context of molecular therapies for muscular dystrophies. Finally, we examine attributes, obstacles, and deployment opportunities for CRISPR-based epigenetic modulating technologies.

Unconstrained Precision Mitochondrial Genome Editing with αDdCBEs.

DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C•G-to-T•A conversions in mitochondrial DNA (mtDNA). DdCBEs work in pairs, with each arm composed of a transcription activator-like effector (TALE), a split double-stranded DNA deaminase half, and a uracil glycosylase inhibitor. This pioneering technology has helped improve our understanding of cellular processes involving mtDNA and has paved the way for the development of models and therapies for genetic disorders caused by pathogenic mtDNA variants. Nonetheless, given the intrinsic properties of TALE proteins, several target sites in human mtDNA are predicted to remain out of reach to DdCBEs and other TALE-based technologies. Specifically, due to the conventional requirement for a thymine immediately upstream of the TALE target sequences (i.e., the 5'-T constraint), over 150 loci in the human mitochondrial genome are presumed to be inaccessible to DdCBEs. Previous attempts at circumventing this requirement, either by developing monomeric DdCBEs or utilizing DNA-binding domains alternative to TALEs, have resulted in suboptimal specificity profiles with reduced therapeutic potential. Here, aiming to challenge and elucidate the relevance of the 5'-T constraint in the context of DdCBE-mediated mtDNA editing, and to expand the range of motifs that are editable by this technology, we generated DdCBEs containing TALE proteins engineered to recognize all 5' bases. These modified DdCBEs are herein referred to as αDdCBEs. Notably, 5'-T-noncompliant canonical DdCBEs efficiently edited mtDNA at diverse loci. However, they were frequently outperformed by αDdCBEs, which exhibited significant improvements in activity and specificity, regardless of the most 5' bases of their TALE binding sites. Furthermore, we showed that αDdCBEs are compatible with the enhanced DddAtox variants DddA6 and DddA11, and we validated TALE shifting with αDdCBEs as an effective approach to optimize base editing outcomes. Overall, αDdCBEs enable efficient, specific, and unconstrained mitochondrial base editing.

Gender and Geographical Representation on Editorial Board Members of Medical Informatics Journals.

Previous work has suggested that gender and geographical distribution (affiliation) of Editors-in-Chief (EiC) and Editorial Board (EB) members are inequitable in representation of scientific communities, and could benefit from increasing diversity of representation. Specifically, previous studies suggest that male and ethnically white (or non-minoritized groups) are overrepresented. Such differences in representation may potentially influence the scientific and scholarly record. This paper aims to build on pre-existing literature by examining the diversity of representation among EiCs and EB members in the top (Q1) journals in the "Medicine-Health Informatics" category (ranked by SCImago Journal and Country Rank, or SJR) in terms of gender as assessed by genderize.io) and geographical distribution of affiliations. Preliminary findings are consistent with those of previous work on the topic: only 25% (8/32) of the EiCs in the selected journals are female, while females only represent 32.7% (426/1303) of the EB members across journals. Furthermore, the US is highly represented in EBs, with more than half of the members, i.e., 52.2% (698/1337), being US-affiliated. Present results suggest the need for an intentional approach to diversifying representation on editorial boards of medical informatics journals. Such intention can be seen as part of a call to action from important stakeholders, including medical informatics leaders and programs, journal management and publishers, and the medical informatics and scientific community more generally.

Knowledge and practices of plagiarism among journal editors of Nepal.

BACKGROUND: This study was conducted to assess the knowledge and ongoing practices of plagiarism among the journal editors of Nepal. METHODS: This web-based questionnaire analytical cross-sectional was conducted among journal editors working across various journals in Nepal. All journal editors from NepJOL-indexed journals in Nepal who provided e-consent were included in the study using a convenience sampling technique. A final set of questionnaires was prepared using Google Forms, including six knowledge questions, three practice questions (with subsets) for authors, and four (with subsets) for editors. These were distributed to journal editors in Nepal via email, Facebook Messenger, Viber, and WhatsApp. Reminders were sent weekly, up to three times. Data analysis was done in R software. Frequencies and percentages were calculated for the demographic variables, correct responses regarding knowledge, and practices related to plagiarism. Independent t-test and one-way ANOVA were used to compare mean knowledge with demographic variables. For all tests, statistical significance was set at p < 0.05. RESULTS: A total of 147 participants completed the survey.The mean age of the participants was found to be 43.61 ± 8.91 years. Nearly all participants were aware of plagiarism, and most had heard of both Turnitin and iThenticate. Slightly more than three-fourths correctly identified that citation and referencing can avoid plagiarism. The overall mean knowledge score was 5.32 ± 0.99, with no significant differences across demographic variables. As authors, 4% admitted to copying sections of others' work without acknowledgment and reusing their own published work without proper citations. Just over one-fifth did not use plagiarism detection software when writing research articles. Fewer than half reported that their journals used authentic plagiarism detection software. Four-fifths of them suspected plagiarism in the manuscripts assigned through their journal. Three out of every five participants reported the plagiarism used in the manuscript to the respective authors. Nearly all participants believe every journal must have plagiarism-detection software. CONCLUSIONS: Although journal editors' knowledge and practices regarding plagiarism appear to be high, they are still not satisfactory. It is strongly recommended to use authentic plagiarism detection software by the journals and editors should be adequately trained and update their knowledge about it.

CRISPR/Cas genome editing in soybean: challenges and new insights to overcome existing bottlenecks.

BACKGROUND: Soybean is a worldwide-cultivated crop due to its applications in the food, feed, and biodiesel industries. Genome editing in soybean began with ZFN and TALEN technologies; however, CRISPR/Cas has emerged and shortly became the preferable approach for soybean genome manipulation since it is more precise, easy to handle, and cost-effective. Recent reports have focused on the conventional Cas9 nuclease, Cas9 nickase (nCas9) derived base editors, and Cas12a (formally Cpf1) as the most commonly used genome editors in soybean. Nonetheless, several challenges in the complex plant genetic engineering pipeline need to be overcome to effectively edit the genome of an elite soybean cultivar. These challenges include (1) optimizing CRISPR cassette design (i.e., gRNA and Cas promoters, gRNA design and testing, number of gRNAs, and binary vector), (2) improving transformation frequency, (3) increasing the editing efficiency ratio of targeted plant cells, and (4) improving soybean crop production. AIM OF REVIEW: This review provides an overview of soybean genome editing using CRISPR/Cas technology, discusses current challenges, and highlights theoretical (insights) and practical suggestions to overcome the existing bottlenecks. KEY SCIENTIFIC CONCEPTS OF REVIEW: The CRISPR/Cas system was discovered as part of the bacterial innate immune system. It has been used as a biotechnological tool for genome editing and efficiently applied in soybean to unveil gene function, improve agronomic traits such as yield and nutritional grain quality, and enhance biotic and abiotic stress tolerance. To date, the efficiency of gRNAs has been validated using protoplasts and hairy root assays, while stable plant transformation relies on Agrobacterium-mediated and particle bombardment methods. Nevertheless, most steps of the CRISPR/Cas workflow require optimizations to achieve a more effective genome editing in soybean plants.

Behind the curtain of Australasian Psychiatry: The practice of a medical journal and a call for reviewers.

The process of medical scientific journal publishing merits further explanation for authors and readers. Prospective authors need to understand the scope of the journal and the article types that are published. We give an overview of the editorial process, including selection of reviewers, peer review and decisions regarding revision, acceptance and rejection of papers for Australasian Psychiatry. We encourage authors and readers to submit papers, and volunteer as peer reviewers, working together with the journal editorial team.

Evaluation of DNA minicircles for delivery of adenine and cytosine base editors using activatable gene on "GO" reporter imaging systems.

Over 30,000 point mutations are associated with debilitating diseases, including many cancer types, underscoring a critical need for targeted genomic solutions. CRISPR base editors, like adenine base editors (ABEs) and cytosine base editors (CBEs), enable precise modifications by converting adenine to guanine and cytosine to thymine, respectively. Challenges in efficiency and safety concerns regarding viral vectors used in delivery limit the scope of base editing. This study introduces non-viral minicircles, bacterial-backbone-free plasmids, as a delivery vehicle for ABEs and CBEs. The research uses cells engineered with the "Gene On" (GO) reporter gene systems for tracking minicircle-delivered ABEs, CBEs, or Cas9 nickase (control), using green fluorescent protein (GFPGO), bioluminescence reporter firefly luciferase (LUCGO), or a highly sensitive Akaluciferase (AkalucGO) designed in this study. The results show that transfection of minicircles expressing CBE or ABE resulted in significantly higher GFP expression and luminescence signals over controls, with minicircles demonstrating the most substantial editing. This study presents minicircles as a new strategy for base editor delivery and develops an enhanced bioluminescence imaging reporter system for tracking ABE activity. Future studies aim to evaluate the use of minicircles in preclinical cancer models, facilitating potential clinical applications.

CRISPR beyond: harnessing compact RNA-guided endonucleases for enhanced genome editing.

The CRISPR-Cas system, an adaptive immunity system in prokaryotes designed to combat phages and foreign nucleic acids, has evolved into a groundbreaking technology enabling gene knockout, large-scale gene insertion, base editing, and nucleic acid detection. Despite its transformative impact, the conventional CRISPR-Cas effectors face a significant hurdle-their size poses challenges in effective delivery into organisms and cells. Recognizing this limitation, the imperative arises for the development of compact and miniature gene editors to propel advancements in gene-editing-related therapies. Two strategies were accepted to develop compact genome editors: harnessing OMEGA (Obligate Mobile Element-guided Activity) systems, or engineering the existing CRISPR-Cas system. In this review, we focus on the advances in miniature genome editors based on both of these strategies. The objective is to unveil unprecedented opportunities in genome editing by embracing smaller, yet highly efficient genome editors, promising a future characterized by enhanced precision and adaptability in the genetic interventions.

Comprehensive analysis of the editing window of C-to-T TALE base editors.

One of the most recent advances in the genome editing field has been the addition of "TALE Base Editors", an innovative platform for cell therapy that relies on the deamination of cytidines within double strand DNA, leading to the formation of an uracil (U) intermediate. These molecular tools are fusions of transcription activator-like effector domains (TALE) for specific DNA sequence binding, split-DddA deaminase halves that will, upon catalytic domain reconstitution, initiate the conversion of a cytosine (C) to a thymine (T), and an uracil glycosylase inhibitor (UGI). We developed a high throughput screening strategy capable to probe key editing parameters in a precisely defined genomic context in cellulo, excluding or minimizing biases arising from different microenvironmental and/or epigenetic contexts. Here we aimed to further explore how target composition and TALEB architecture will impact the editing outcomes. We demonstrated how the nature of the linker between TALE array and split DddAtox head allows us to fine tune the editing window, also controlling possible bystander activity. Furthermore, we showed that both the TALEB architecture and spacer length separating the two TALE DNA binding regions impact the target TC editing dependence by the surrounding bases, leading to more restrictive or permissive editing profiles.

Gender Parity Analysis of the Editorial Boards of Influential Dermatology Journals: Cross-Sectional Study.

This study underscores the persistent underrepresentation of women in academic dermatology leadership positions by examining the gender composition of editorial boards across top dermatology journals, emphasizing the urgent need for proactive strategies to promote diversity, equity, and inclusion.

DNA base editing corrects common hemophilia A mutations and restores factor VIII expression in in vitro and ex vivo models.

BACKGROUND: Replacement and nonreplacement therapies effectively control bleeding in hemophilia A (HA) but imply lifelong interventions. Authorized gene addition therapy could provide a cure but still poses questions on durability. FVIIIgene correction would definitively restore factor (F)VIII production, as shown in animal models through nuclease-mediated homologous recombination (HR). However, low efficiency and potential off-target double-strand break still limit HR translatability. OBJECTIVES: To correct common model single point mutations leading to severe HA through the recently developed double-strand break/HR-independent base editing (BE) and prime editing (PE) approaches. METHODS: Screening for efficacy of BE/PE systems in HEK293T cells transiently expressing FVIII variants and validation at DNA (sequencing) and protein (enzyme-linked immunosorbent assay; activated partial thromboplastin time) level in stable clones. Evaluation of rescue in engineered blood outgrowth endothelial cells by lentiviral-mediated delivery of BE. RESULTS: Transient assays identified the best-performing BE/PE systems for each variant, with the highest rescue of FVIII expression (up to 25% of wild-type recombinant FVIII) for the p.R2166∗ and p.R2228Q mutations. In stable clones, we demonstrated that the mutation reversion on DNA (∼24%) was consistent with the rescue of FVIII secretion and activity of 20% to 30%. The lentiviral-mediated delivery of the selected BE systems was attempted in engineered blood outgrowth endothelial cells harboring the p.R2166∗ and p.R2228Q variants, which led to an appreciable and dose-dependent rescue of secreted functional FVIII. CONCLUSION: Overall data provide the first proof-of-concept for effective BE/PE-mediated correction of HA-causing mutations, which encourage studies in mouse models to develop a personalized cure for large cohorts of patients through a single intervention.