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Editing the RR-TZF Gene Subfamily in Rice Uncovers Potential Risks of CRISPR/Cas9 for Targeted Genetic Modification.
The CRISPR/Cas9 system offers a powerful tool for gene editing to enhance rice productivity. In this study, we successfully edited eight RR-TZF genes in japonica rice Nipponbare using CRISPR-Cas9 technology, achieving a high editing efficiency of 73.8%. Sequencing revealed predominantly short insertions or deletions near the PAM sequence, along with multi-base deletions often flanked by identical bases. Off-target analysis identified 5 out of 31 predicted sites, suggesting the potential for off-target effects, which can be mitigated by designing gRNAs with more than three base mismatches. Notably, new mutations emerged in the progeny of several gene-edited mutants, indicating inheritable genetic mutagenicity. Phenotypic analysis of homozygous mutants revealed varied agronomic traits, even within the same gene, highlighting the complexity of gene-editing outcomes. These findings underscore the importance of backcrossing to minimize off-target and inheritable mutagenicity effects, ensuring more accurate trait evaluation. This study offers insights into CRISPR/Cas9 mechanisms and uncertain factors and may inform future strategies for rice improvement, prompting further research into CRISPR/Cas9's precision and long-term impacts.
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Cytogenetic Analysis of the Results of Genome Editing on the Cell Model of Parkinson's Disease.
We performed a cytogenetic analysis of the results of CRISPR/Cas9-correction of G2019S mutation in LRRK2 gene associated with Parkinson's disease. Genome editing was performed on induced pluripotent stem cells derived from fibroblasts of a patient carrying this mutation. A mosaic variant of tetraploidy 92 XXYY/46,XY (24-43% cells from various clones) was found in neuronal precursors differentiated from the induced pluripotent stem cells after gene editing procedure. Solitary cases of translocations and chromosome breaks were observed. These data confirm the importance of the development of new approaches ensuring genome stability in CRISPR/Cas9-edited cultures.
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Base editors hit the clinic.
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Addressing the Blind Spots of Deaminase Base Editors with CRISPR-Cas Glycosylase-Based Editors.
The emergence of CRISPR base editors signifies a pivotal shift in genome editing, moving beyond the "cut-and-paste" approach into an era of precise "chemical rewriting." While early editors including CBEs and ABEs enabled efficient base transitions by combining deaminases with Cas9 nickase, their core mechanism limited their application to transition mutations, leaving nearly half of disease-causing transversions unresolved. This review outlines the transformation of DNA glycosylases from unwanted side-effect generators into core drivers of base editor evolution. Through strategic protein engineering, glycosylases have been harnessed to create novel editors that mediate crucial transversion edits, substantially expanding the targeting landscape of precision genome editing. We will examine how these engineered glycosylases achieve programmable DNA rewriting through redirected DNA repair pathways, compare emerging and conventional editors, and explore applications in genetic therapy and functional genomics while addressing ongoing challenges in specificity and delivery. Finally, we envision next-generation editors equipped with artificial intelligence-designed glycosylases capable of spatiotemporally controlled genome manipulation.
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Comprehensive evaluation and prediction of editing outcomes for near-PAMless adenine and cytosine base editors.
Base editors enable the direct conversion of target bases without inducing double-strand breaks, showing great potential for disease modeling and gene therapy. Yet, their applicability has been constrained by the necessity for specific protospacer adjacent motif (PAM). We generate four versions of near-PAMless base editors and systematically evaluate their editing patterns and efficiencies using an sgRNA-target library of 45,747 sequences. Near-PAMless base editors significantly expanded the targeting scope, with both PAM and target flanking sequences as determinants for editing outcomes. We develop BEguider, a deep learning model, to accurately predict editing results for near-PAMless base editors. We also provide experimentally measured editing outcomes of 20,541 ClinVar sites, demonstrating that variants previously inaccessible by NGG PAM base editors can now be precisely generated or corrected. We make our predictive tool and data available online to facilitate development and application of near-PAMless base editors in both research and clinical settings.
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Mitochondrial genome editing tools: prospects in animal breeding.
Mitochondria are vital organelles responsible for driving cellular energy metabolism and regulating key biological processes. Their circular mitochondrial DNA (mtDNA) encodes 13 subunits of the respiratory chain proteins but is susceptible to mutations due to high levels of reactive oxygen species and limited repair mechanisms. Mutant phenotypes manifest only when heteroplasmy surpasses a critical threshold. Understanding the consequences of mtDNA mutations has long been hampered by the lack of precise editing tools. Recently, CRISPR-free, protein-only mitochondrial base editors have enabled C·G-to-T·A and A·T-to-G·C transitions. These breakthroughs facilitate the creation of relevant disease models and offer unique opportunities for animal breeding, as specific mtDNA variants are known to influence economically important traits in livestock, including production, reproduction, and stress tolerance. This review summarizes recent advances in mitochondrial genome editing technologies, including CRISPR/Cas-based systems, restriction endonucleases, double-stranded DNA deaminase toxin A (DddA)-based cytosine and adenine base editors, and DddA-free base editors, along with their delivery strategies and optimization avenues. Furthermore, we outline the associations between mtDNA polymorphisms, copy number variation, and economic traits in livestock and poultry. Finally, we discuss the potential applications of mitochondrial genome editing in animal breeding and highlight the critical safety and ethical considerations that require careful attention.
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Efficient genome editing in the olive fruit fly, Bactrocera oleae.
The olive fruit fly, Bactrocera oleae, causes great damage to the quality and quantity of olive production worldwide. Pest management approaches have proved difficult for a variety of reasons, a fact that has brought about a need for alternative tools and approaches. Here we report for the first time in B. oleae the development of the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene editing tool, using the well-known eye colour marker gene scarlet. Two synthetic guide RNAs targeting the coding region of the scarlet gene were synthesized and shown to work efficiently in vitro. These reagents were then microinjected along with purified Cas9 protein into early-stage embryos. Successful CRISPR-induced mutations of both copies of the scarlet gene showed a striking yellow eye phenotype, indicative of gene disruption. Multiple successful CRISPR events were confirmed by PCR and sequencing. The establishment of an efficient CRISPR-based gene editing tool in B. oleae will enable the study of critical molecular mechanisms in olive fruit fly biology and physiology, including the analysis of insecticide resistance mechanisms and the discovery of novel insecticide targets, as well as facilitate the development of novel biotechnology-based pest control strategies.
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Diverse Class 2 CRISPR Effectors as Active Nucleases with Expanded Targeting Capabilities.
CRISPR-Cas systems have proven effective in a variety of applications due to their ease of use and relatively high editing efficiency. Yet, any individual CRISPR-Cas system has inherent limitations, necessitating a diversity of RNA-guided nucleases to suit applications with distinct needs. We searched through metagenomic sequences to identify RNA-guided nucleases and found enzymes from diverse CRISPR-Cas types and subtypes, the most promising of which we developed into gene-editing platforms. Based on prior annotations of the metagenomic sequences, we establish the likely taxa and sampling locations where Class 2 CRISPR-Cas systems active in eukaryotes may be found. The newly discovered systems show robust capabilities as gene editors and base editors.
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Fast and Quantitative Identification of Ex Vivo Precise Genome Targeting-Induced Indel Events by IDAA.
Recent developments in gene targeting methodologies such as ZFNs, TALENs, and CRISPR/Cas9 have revolutionized approaches for gene modifications in cells, tissues, and whole animals showing great promise for translational applications. With regard to CRISPR/Cas9, a variety of repurposed systems have been developed to achieve gene knock-out, base editing, targeted knock-in, gene activation/repression, epigenetic modulation, and locus-specific labeling. A functional communality of all CRISPR/Cas9 applications is the gRNA-dependent targeting specificity of the Cas9/gRNA complex that, for gene knock-out (KO) purposes, has been shown to dictate the indel formation potential. Therefore, the objective of a CRISPR/Cas9 KO set up is to identify gRNA designs that enable maximum out-of-frame insertion and/or deletion (indel) formation and thus, gRNA design becomes a proxy for optimal functionality of CRISPR/Cas9 KO and repurposed systems. To this end, validation of gRNA functionality depends on efficient, accurate, and sensitive identification of indels induced by a given gRNA design. For in vitro indel profiling the most commonly used methods are based on amplicon size discrimination or sequencing. Indel detection by amplicon analysis (IDAA™) is an alternative sensitive, fast, and cost-efficient approach ideally suited for profiling of indels induced by Cas9/gRNA with similar sensitivity, specificity, and resolution, down to single base discrimination, as the preferred next-generation sequencing-based indel profiling methodologies. Here we provide a protocol that is based on complexed Cas9/gRNA RNPs delivered to primary peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals followed by quantitative IDAA indel profiling. Importantly, the protocol described benefits from a short "sample-to-data" turnaround time of less than 5 h. Thus, this protocol describes a methodology that provides a suitable and effective solution to validate and quantify the extent of ex vivo CRISPR/Cas9 targeting in primary cells.
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Enhanced Bombyx genome editing via Cas9 ribonucleoprotein injection.
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