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Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system.
Precise genetic modifications in model animals are essential for biomedical research. Here, we report a programmable "base editing" system to induce precise base conversion with high efficiency in zebrafish. Using cytidine deaminase fused to Cas9 nickase, up to 28% of site-specific single-base mutations are achieved in multiple gene loci. In addition, an engineered Cas9-VQR variant with 5'-NGA PAM specificities is used to induce base conversion in zebrafish. This shows that Cas9 variants can be used to expand the utility of this technology. Collectively, the targeted base editing system represents a strategy for precise and effective genome editing in zebrafish.The use of base editing enables precise genetic modifications in model animals. Here the authors show high efficient single-base editing in zebrafish using modified Cas9 and its VQR variant with an altered PAM specificity.
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Cutting a path to effective delivery of genome engineering machinery.
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BEAT: A Python Program to Quantify Base Editing from Sanger Sequencing.
Through fusing CRISPR-Cas9 nickases with cytidine or adenine deaminases, a new paradigm-shifting class of genome-editing technology, termed "base editors," has recently been developed. Base editors mediate highly efficient, targeted single-base conversion without introducing double-stranded breaks. Analysis of base editing outcomes typically relies on imprecise enzymatic mismatch cleavage assays, time-consuming single-colony sequencing, or expensive next-generation deep sequencing. To overcome these limitations, several groups have recently developed computer programs to measure base-editing efficiency from fluorescence-based Sanger sequencing data such as Edit deconvolution by inference of traces in R (EditR), TIDER, and ICE. These approaches have greatly simplified the quantitation of base-editing experiments. However, the current Sanger sequencing tools lack the capability of batch analysis and producing high-quality images for publication. Here, we provide a base editing analysis tool (BEAT) written in Python to analyze and quantify the base-editing events from Sanger sequencing data in a batch manner, which can also produce intuitive, publication-ready base-editing images.
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Multiplex Gene Disruption by Targeted Base Editing of Yarrowia lipolytica Genome Using Cytidine Deaminase Combined with the CRISPR/Cas9 System.
The oleaginous yeast Yarrowia lipolytica has a tendency to use the non-homologous end joining repair (NHEJ) over the homology directed recombination as double-strand breaks (DSB) repair system, making it difficult to edit the genome using homologous recombination. A recently developed Target-AID (activation-induced cytidine deaminase) base editor, designed to recruit cytidine deaminase (CDA) to the target DNA locus via the CRISPR/Cas9 system, can directly induce C to T mutation without DSB and donor DNA. In this study, this system is adopted in Y. lipolytica for multiplex gene disruption. Target-specific gRNA(s) and a fusion protein consisting of a nickase Cas9, pmCDA1, and uracil DNA glycosylase inhibitor are expressed from a single plasmid to disrupt target genes by introducing a stop codon via C to T mutation within the mutational window. Deletion of the KU70 gene involved in the NHEJ prevents the generation of indels by base excision repair following cytidine deamination, increasing the accuracy of genome editing. Using this Target-AID system with optimized expression levels of the base editor, single gene disruption and simultaneous double gene disruption are achieved with the efficiencies up to 94% and 31%, respectively, demonstrating this base editing system as a convenient genome editing tool in Y. lipolytica.
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Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing.
Gene editing nucleases such as CRISPR/Cas9 have enabled efficient and precise gene editing in vitro and hold promise of eventually achieving in vivo gene editing based therapy. However, a major challenge for their use is the lack of a safe and effective virus-free system to deliver gene editing nuclease elements. Polymers are a promising class of delivery vehicle due to their higher safety compared to currently used viral vectors, but polymers suffer from lower transfection efficiency. Polymeric vectors have been used for small nucleotide delivery but have yet to be used successfully with plasmid DNA (pDNA), which is often several hundred times larger than small nucleotides, presenting an engineering challenge. To address this, we extended our previously reported hyperbranched polymer (HP) delivery system for pDNA delivery by synthesizing several variants of HPs: HP-800, HP-1.8K, HP-10K, HP-25K. We demonstrate that all HPs have low toxicity in various cultured cells, with HP-25K being the most efficient at packaging and delivering pDNA. Importantly, HP-25K mediated delivery of CRISPR/Cas9 pDNA resulted in higher gene-editing rates than all other HPs and Lipofectamine at several clinically significant loci in different cell types. Consistently, HP-25K also led to more robust base editing when delivering the CRISPR base editor "BE4-max" pDNA to cells compared with Lipofectamine. The present work demonstrates that HP nanoparticles represent a promising class of vehicle for the non-viral delivery of pDNA towards the clinical application of gene-editing therapy.
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CRISPR-based knockout and base editing confirm the role of MYRF in heart development and congenital heart disease.
High-throughput DNA sequencing studies increasingly associate DNA variants with congenital heart disease (CHD). However, functional modeling is a crucial prerequisite for translating genomic data into clinical care. We used CRISPR-Cas9-mediated targeting of 12 candidate genes in the vertebrate model medaka (Oryzias latipes), five of which displayed a novel cardiovascular phenotype spectrum in F0 (crispants): mapre2, smg7, cdc42bpab, ankrd11 and myrf, encoding a transcription factor recently linked to cardiac-urogenital syndrome. Our myrf mutant line showed particularly prominent embryonic cardiac defects recapitulating phenotypes of pediatric patients, including hypoplastic ventricle. Mimicking human mutations, we edited three sites to generate specific myrf single-nucleotide variants via cytosine and adenine base editors. The Glu749Lys missense mutation in the conserved intramolecular chaperon autocleavage domain fully recapitulated the characteristic myrf mutant phenotype with high penetrance, underlining the crucial function of this protein domain. The efficiency and scalability of base editing to model specific point mutations accelerate gene validation studies and the generation of human-relevant disease models.
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Effects of Different Gene Editing Modes of CRISPR/Cas9 on Soybean Fatty Acid Anabolic Metabolism Based on GmFAD2 Family.
Δ12-fatty acid dehydrogenase (FAD2) is the essential enzyme responsible for catalyzing the formation of linoleic acid from oleic acid. CRISPR/Cas9 gene editing technology has been an essential tool for molecular breeding in soybeans. To evaluate the most suitable type of gene editing in soybean fatty acid synthesis metabolism, this study selected five crucial enzyme genes of the soybean FAD2 gene family-GmFAD2-1A, GmFAD2-1B, GmFAD2-2A, GmFAD2-2B, and GmFAD2-2C-and created a CRISPR/Cas9-mediated single gene editing vector system. The results of Sanger sequencing showed that 72 transformed plants positive for T1 generation were obtained using Agrobacterium-mediated transformation, of which 43 were correctly edited plants, with the highest editing efficiency of 88% for GmFAD2-2A. The phenotypic analysis revealed that the oleic acid content of the progeny of GmFAD2-1A gene-edited plants had a higher increase of 91.49% when compared to the control JN18, and the rest of the gene-edited plants in order were GmFAD2-2A, GmFAD2-1B, GmFAD2-2C, and GmFAD2-2B. The analysis of gene editing type has indicated that base deletions greater than 2bp were the predominant editing type in all editing events. This study provides ideas for the optimization of CRISPR/Cas9 gene editing technology and the development of new tools for precise base editing in the future.
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Recent Advances in CRISPR/Cas9-Based Genome Editing Tools for Cardiac Diseases.
In the past two decades, genome editing has proven its value as a powerful tool for modeling or even treating numerous diseases. After the development of protein-guided systems such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), which for the first time made DNA editing an actual possibility, the advent of RNA-guided techniques has brought about an epochal change. Based on a bacterial anti-phage system, the CRISPR/Cas9 approach has provided a flexible and adaptable DNA-editing system that has been able to overcome several limitations associated with earlier methods, rapidly becoming the most common tool for both disease modeling and therapeutic studies. More recently, two novel CRISPR/Cas9-derived tools, namely base editing and prime editing, have further widened the range and accuracy of achievable genomic modifications. This review aims to provide an overview of the most recent developments in the genome-editing field and their applications in biomedical research, with a particular focus on models for the study and treatment of cardiac diseases.
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Gene Therapy Techniques and Delivery Methods (Review).
Gene therapy has evolved into a sophisticated field encompassing diverse precision editing platforms and advanced delivery systems capable of addressing complex genetic disorders and age-related pathologies. This comprehensive review examines the current landscape of gene therapeutic technologies, including CRISPR-based genome editing, base editing systems, prime editing platforms, and emerging DNA polymerase-based editors alongside their corresponding delivery methodologies. The review encompasses viral vectors, including tissue-specific adeno-associated virus serotypes, non-viral delivery systems such as ionizable lipid nanoparticles and virus-like particles, and innovative platforms, including exosome-based delivery and the SEND system. We examine therapeutic applications spanning nuclear genome editing, mitochondrial genome modification, RNA editing, and epigenetic modulation, demonstrating the expanding scope of gene therapy beyond traditional monogenic disorders. Critical analysis reveals that while fundamental technological capabilities have been established, significant challenges remain in manufacturing scalability, long-term safety assessment, delivery across physiological barriers, and optimization of editing efficiency in post-mitotic tissues. The integration of artificial intelligence approaches for predictive analysis and rational vector design represents a promising avenue for addressing current limitations. This review concludes that successful clinical implementation requires systematic resolution of manufacturing, safety, and delivery challenges alongside the development of standardized protocols for patient stratification and robust regulatory frameworks that accommodate rapid technological innovation while ensuring patient safety.
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Engineering IscB to develop highly efficient miniature editing tools in mammalian cells and embryos.
The IscB proteins, as the ancestors of Cas9 endonuclease, hold great promise due to their small size and potential for diverse genome editing. However, their activity in mammalian cells is unsatisfactory. By introducing three residual substitutions in IscB, we observed an average 7.5-fold increase in activity. Through fusing a sequence-non-specific DNA-binding protein domain, the eIscB-D variant achieved higher editing efficiency, with a maximum of 91.3%. Moreover, engineered ωRNA was generated with a 20% reduction in length and slightly increased efficiency. The engineered eIscB-D/eωRNA system showed an average 20.2-fold increase in activity compared with the original IscB. Furthermore, we successfully adapted eIscB-D for highly efficient cytosine and adenine base editing. Notably, eIscB-D is highly active in mouse cell lines and embryos, enabling the efficient generation of disease models through mRNA/ωRNA injection. Our study suggests that these miniature genome-editing tools have great potential for diverse applications.
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