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Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses.
Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for precise gene editing in plants. However, simultaneous gene editing of multiple homoeoalleles remains challenging, especially in self-incompatible polyploid plants. Here, we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue, using both CRISPR/Cas9 and LbCas12a (LbCpf1) systems. Loss-of-function mutants of FaPDS exhibited albino leaves, while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue. Moreover, these mutations were inheritable. Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.
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Dissecting Plant Gene Functions Using CRISPR Toolsets for Crop Improvement.
The CRISPR-based gene editing technology has become more and more powerful in genome manipulation for agricultural breeding, with numerous improved toolsets springing up. In recent years, many CRISPR toolsets for gene editing, such as base editors (BEs), CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), and plant epigenetic editors (PEEs), have been developed to clarify gene function and full-level gene regulation. Here, we comprehensively summarize the application and capacity of the different CRISPR toolsets in the study of plant gene expression regulation, highlighting their potential application in gene regulatory networks' analysis. The general problems in CRISPR application and the optimal solutions in the existing schemes for high-throughput gene function analysis are also discussed. The CRISPR toolsets targeting gene manipulation discussed here provide new solutions for further genetic improvement and molecular breeding of crops.
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CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion.
BACKGROUND: Halophiles possess several unique properties and have broad biotechnological applications including industrial biotechnology production. Halomonas spp., especially Halomonas bluephagenesis, have been engineered to produce various biopolyesters such as polyhydroxyalkanoates (PHA), some proteins, small molecular compounds, organic acids, and has the potential to become a chassis cell for the next-generation of industrial biotechnology (NGIB) owing to its simple culture, fast growth, contamination-resistant, low production cost, and high production value. An efficient genome editing system is the key for its engineering and application. However, the efficiency of the established CRISPR-Cas-homologous recombination (HR) gene editing tool for large DNA fragments was still relatively low. In this study, we firstly report a CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas bluephagenesis. RESULTS: Three different NHEJ repair systems were selected and functionally identified in Halomonas bluephagenesis TD01. The NHEJ system from M. tuberculosis H37Rv (Mt-NHEJ) can functionally work in H. bluephagenesis TD01, resulting in base deletion of different lengths for different genes and some random base insertions. Factors affecting knockout efficiencies, such as the number and position of sgRNAs on the DNA double-strands, the Cas9 protein promoter, and the interaction between the HR and the NHEJ repair system, were further investigated. Finally, the optimized CRISPR-Cas9-NHEJ editing system was able to delete DNA fragments up to 50 kb rapidly with high efficiency of 31.3%, when three sgRNAs on the Crick/Watson/Watson DNA double-strands and the arabinose-induced promoter Para for Cas9 were used, along with the background expression of the HR repair system. CONCLUSIONS: This was the first report of CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas spp. These results not only suggest that this editing system is a powerful genome engineering tool for constructing chassis cells in Halomonas, but also extend the application of the NHEJ repair system.
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Therapeutic Gene Editing in Inherited Retinal Disorders.
Since the development of CRISPR/Cas9 gene editing in 2012, therapeutic editing research has produced several phase 1-2a trials. Here we provide an overview of the mechanisms and applications of various gene-editing technologies including adeno-associated virus vectors, lentiviruses, CRISPR/Cas9 systems, base and prime editing, antisense oligonucleotides, short-hairpin RNAs, Cas13, and adenosine deaminase acting on RNA for the treatment of various inherited retinal diseases (IRDs). We outline the various stages of clinical trials using these technologies and the impacts they have made in advancing the practice of medicine.
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Profiling Genome-Wide Specificity of dCpf1 Cytidine Base Editors Using Digenome-Seq.
Digenome-seq is a powerful approach for determining the genome-wide specificity of programmable nuclease including CRISPR-Cas9 and CRISPR-Cpf1 (also known as Cas12a) and programmable deaminase including cytosine base editors (CBEs) and adenine base editors (ABEs). To define the genome-wide specificity of dLbCpf1-BE (also known as dLbCas12a-BE), genomic DNA is first incubated with dLbCpf1-BE, which induces C-to-U conversion at on-target and off-target sites, and then treated with a mixture of E. coli uracil DNA glycosylase (UDG) and Endonuclease VIII, which creates single-strand breaks (SSBs) by removing uracil in vitro. Digested genomic DNA is subjected to WGS, and then sequencing reads are aligned to the reference genome, resulting in straight alignments at on-target and off-target sites. The in vitro cleavage sites related to the straight alignments can be identified using the Digenome-seq computer tool.
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Compact 9dBEs Enable Efficient and Precise Genome Editing in Mammalian Cells and In Vivo.
Despite the promise of DNA base editors for diverse genome editing applications, their utility remains constrained by off-target effects, which are exacerbated by short spacers in miniature systems and the large size of Cas9-derived editors, which impedes adeno-associated virus (AAV) delivery. Here, guided by structural insights into a compact Cas9d nuclease from Deltaproteobacteria, we developed an efficient Cas9d system (Cas9dUltra) through gRNA and protein engineering, and further developed its base editors (9dBEs). Cas9dUltra and 9dBEs enabled efficient and precise genome editing in human cells. Notably, 9dCBE induced premature termination codons in 89% of mouse pups by microinjection, facilitating robust disease modeling. Furthermore, a single AAV delivering 9dCBE achieved efficient Pcsk9 editing and a concomitant reduction in serum LDL-C levels in mice. Collectively, this study establishes a series of compact, potent genome editing tools poised to advance biological and biomedical translational research.
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Gene Therapy and Gene Editing in Type 1 Diabetes: CRISPR-Based β-Cell Replacement and Treg Immune Modulation Approaches.
BACKGROUND: Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease marked by the destruction of pancreatic β-cells, resulting in lifelong dependence on exogenous insulin. Despite advances in insulin delivery and glucose monitoring technologies, patients remain at risk for acute and long-term complications, underscoring the need for curative strategies. Gene therapy and gene-editing technologies are emerging as transformative approaches capable of restoring β-cell function, modulating immune responses and potentially achieving durable remission. METHOD: This review synthesizes basic science foundations and clinical trial evidence, focusing on five key protocols (NCT03162237, NCT05210530, NCT05241444, NCT05565248 and NCT06938334). RESULTS: Strategies include immune modulation (PD-L1, FOXP3), β-cell replacement (CRISPR-edited progenitors, xenotransplantation) and combination approaches. Early-phase clinical trials have demonstrated feasibility and safety; however, long-term efficacy, durability and scalability remain uncertain. Critical challenges include potential off-target effects in CRISPR editing, risks of insertional mutagenesis, safety concerns in xenotransplantation and achieving a balance between immune tolerance and protective immunity. Future directions emphasize combination therapies, personalized medicine and next-generation editing tools such as base and prime editing. CONCLUSION: Together, these efforts represent a paradigm shift from symptomatic insulin replacement toward curative interventions, while highlighting the considerable translational hurdles that must be overcome before routine clinical application.
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Editing base in mouse model.
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CRISPR/Cas9-mediated editing of the GhJAZ2 gene improves fiber length and lint percentage in Gossypium hirsutum L.
Cotton is regarded as a strategic agricultural commodity owing to its renewable and naturally derived fiber. With the escalating global demand for high-quality fiber, genetic improvement of fiber traits is a critical focus for sustaining and advancing the textile industry standards. The cotton GhJAZ2 gene encodes the Jasmonate ZIM-domain 2 protein, a known repressor in the jasmonic acid signaling pathway and negatively regulates fiber initiation. In this study, we designed a gRNA that simultaneously targets GhJAZ2 homologs and assembled it into the CRISPR vector (pHSE401). Subsequently, the construct (pHSE401-gRNA) was transformed into cotton (Gossypium hirsutum L.) using an Agrobacterium-mediated in planta transformation strategy, targeting the shoot apical meristem as the primary site of transformation. Sanger sequencing analysis revealed consistent single-base pair indels at the targeted site across both A and D sub-genomes, with edited T1 progenies showing both inherited and newly introduced indels at the targeted loci. Fiber analysis of edited lines compared to the control revealed a significant (p < .05) enhancement in lint percentage (≤13.74%) and fiber length (≤16.91%). This study demonstrated the effective application of CRISPR/Cas9 for targeted trait improvement in cotton, offering GhJAZ2-edited lines that can be advanced to develop transgene-free cultivars with improved fiber traits.
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Base Editors and Off-Targeting: The Deaminase Matters.
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