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Special Issue: Latest Research on Plant Genomics and Genome Editing.
Over the past ten years, plant science has undergone a remarkable transformation driven by the convergence of next-generation sequencing, increasingly sophisticated bioinformatics tools, and the rise of targeted genome editing platforms [...].
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May I Cut in? Gene Editing Approaches in Human Induced Pluripotent Stem Cells.
In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies have further accelerated the potential of hiPSCs. In this review, we discuss the various gene editing technologies that are being implemented with hiPSCs. Specifically, we describe the emergence of technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 that can be used to edit the genome at precise locations, and discuss the strengths and weaknesses of each of these technologies. In addition, we present the current applications of these technologies in elucidating the mechanisms of human development and disease, developing novel and effective therapeutic molecules, and engineering cell-based therapies. Finally, we discuss the emerging technological advances in targeted gene editing methods.
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Enhanced CRISPR-Cas9 RNA system delivery using cell penetrating peptides-based nanoparticles for efficient in vitro and in vivo applications.
CRISPR-Cas9 system has emerged as a revolutionary gene-editing tool with huge therapeutic potential for addressing the underlying genetic causes of various diseases, including cancer. However, there are challenges such as the delivery method that must be overcome for its clinical application. In addition to the risk of nuclease degradation and rapid clearance of the CRISPR-Cas9 system by macrophages, the large size of Cas9, the high anionic charge density and hydrophilic nature of the RNA hinder their intracellular delivery and overall gene transfection efficiency. In this study, we engineered a novel Peptide-Based Nanoparticles ADGN for the delivery of long RNA. ADGN peptides can form stable self-assembled nanoparticles with CRISPR-Cas9 RNA. They have the ability to cross the cell membrane of various cell types, exhibiting a preference for cancer cells that overexpress laminin receptor and safeguard RNA prior their delivery into the cytoplasm. We demonstrate that ADGN peptides significantly promote CRISPR-Cas9 mediated knockout of the luciferase gene in vitro achieving 60 % efficiency with a preference for G insertion at the targeted site of luciferase gene. Moreover, we have provided evidence that these nanoparticles can also be systemically intravenously administrated in vivo in mice to deliver a functional CRISPR-Cas9 system to tumoral lung cells orthotopically implanted in the mouse, resulting in an effective gene knockout in mice. We also demonstrated that the in vivo distribution of ADGN-RNA is influenced by its peptides to RNA molar ratio. This study introduces a promising new Peptide-Based Nanoparticles for delivering CRISPR-Cas9 system in its RNA form applicable in both in vitro and in vivo models.
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PEAC-seq adopts Prime Editor to detect CRISPR off-target and DNA translocation.
CRISPR technology holds significant promise for biological studies and gene therapies because of its high flexibility and efficiency when applied in mammalian cells. But endonuclease (e.g., Cas9) potentially generates undesired edits; thus, there is an urgent need to comprehensively identify off-target sites so that the genotoxicities can be accurately assessed. To date, it is still challenging to streamline the entire process to specifically label and efficiently enrich the cleavage sites from unknown genomic locations. Here we develop PEAC-seq, in which we adopt the Prime Editor to insert a sequence-optimized tag to the editing sites and enrich the tagged regions with site-specific primers for high throughput sequencing. Moreover, we demonstrate that PEAC-seq could identify DNA translocations, which are more genotoxic but usually overlooked by other off-target detection methods. As PEAC-seq does not rely on exogenous oligodeoxynucleotides to label the editing site, we also conduct in vivo off-target identification as proof of concept. In summary, PEAC-seq provides a comprehensive and streamlined strategy to identify CRISPR off-targeting sites in vitro and in vivo, as well as DNA translocation events. This technique further diversified the toolkit to evaluate the genotoxicity of CRISPR applications in research and clinics.
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TIGR-Tas and the Expanding Universe of RNA-Guided Genome Editing Systems: A New Era Beyond CRISPR-Cas.
The recent discovery of TIGR-Tas (Tandem Interspaced Guide RNA-Targeting Systems) marks a major advance in the field of genome editing, introducing a new class of compact, programmable DNA-targeting systems that function independently of traditional CRISPR-Cas pathways. TIGR-Tas effectors use a novel dual-spacer guide RNA (tigRNA) to recognize both strands of target DNA without requiring a protospacer adjacent motif (PAM). These Tas proteins introduce double-stranded DNA cuts with characteristic 8-nucleotide 3' overhangs and are significantly smaller than Cas9, offering delivery advantages for in vivo editing. Structural analyses reveal homology to box C/D snoRNP proteins, suggesting a previously unrecognized evolutionary lineage of RNA-guided nucleases. This review positions TIGR-Tas at the forefront of a new wave of RNA-programmable genome-editing technologies. In parallel, I provide comparative insight into the diverse and increasingly modular CRISPR-Cas systems, including Cas9, Cas12, Cas13, and emerging effectors like Cas3, Cas10, CasΦ, and Cas14. While the CRISPR-Cas universe has revolutionized molecular biology, TIGR-Tas systems open a complementary and potentially more versatile path for programmable genome manipulation. I discuss mechanistic distinctions, evolutionary implications, and potential applications in human cells, synthetic biology, and therapeutic genome engineering.
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Protocol for efficient CRISPR/AAV-mediated genome editing and erythroid differentiation of human hematopoietic stem and progenitor cells.
Here, we present a protocol for genome editing in human hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)-mediated homology-directed repair. We describe steps for AAV production, purification, and titration; HSPC thawing and culture; genome editing; and quantification of editing frequencies. We then detail procedures for erythroid differentiation assays. This protocol ensures high editing efficiency while maintaining cell viability and engraftment potential. For complete details on the use and execution of this protocol, please refer to Chu et al.1.
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CRISPR-/Cas9-Mediated Precise and Efficient Genome Editing in Drosophila.
The CRISPR/Cas9 system provides the means to make precise and purposeful modifications to the genome via homology-directed repair (HDR). In Drosophila, a wide variety of tools provide flexibility to achieve these ends. Here, we detail a method to generate precise genome edits via HDR that is efficient and broadly applicable to any Drosophila stock or species. sgRNAs are first tested for their cleavage efficiency by injecting embryos with Cas9/sgRNA ribonucleoproteins using commercially available Cas9 protein. Using an empirically validated sgRNA, HDR is performed using a donor repair plasmid that carries two transformation markers. A fluorescent eye marker that can be seamlessly removed using PiggyBac transposase marks integration of the repair sequence. A counter-selection marker that produces small rough eyes via RNAi against eyes absent is used to screen against imprecise HDR events. Altogether, the enhancements implemented in this method expand the ease and scope of achieving precise CRISPR/Cas9 genome edits in Drosophila.
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Molecular circuits for genomic recording of cellular events.
Advances in precise genome editing are enabling genomic recordings of cellular events. Since the initial demonstration of CRISPR-based genome editing, the field of genomic recording has witnessed key strides in lineage recording, where clonal lineage relationships among cells are indirectly recorded as synthetic mutations. However, methods for directly recording and reconstructing past cellular events are still limited, and their potential for revealing new insights into cell fate decisions has yet to be realized. The field needs new sensing modules and genetic circuit architectures that faithfully encode past cellular states into genomic DNA recordings to achieve such goals. Here we review recently developed strategies to construct diverse sensors and explore how emerging synthetic biology tools may help to build molecular circuits for genomic recording of diverse cellular events.
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Comparative targeting analysis of KLF1, BCL11A, and HBG1/2 in CD34+ HSPCs by CRISPR/Cas9 for the induction of fetal hemoglobin.
β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB). Imbalanced expression of adult hemoglobin (HbA) induces strong anemia in patients suffering from the disease. However, individuals with natural-occurring mutations in the HBB cluster or related genes, compensate this disparity through γ-globin expression and subsequent fetal hemoglobin (HbF) production. Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). In this study, we thoroughly compared the different CRISPR/Cas9 gene-disruption strategies by gene editing analysis and assessed their safety profile by RNA-seq and GUIDE-seq. All approaches reached therapeutic levels of HbF after gene editing and showed similar gene expression to the control sample, while no significant off-targets were detected by GUIDE-seq. Likewise, all three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices. Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of β-hemoglobinopathies.
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Advances in the delivery of RNA therapeutics: from concept to clinical reality.
The rapid expansion of the available genomic data continues to greatly impact biomedical science and medicine. Fulfilling the clinical potential of genetic discoveries requires the development of therapeutics that can specifically modulate the expression of disease-relevant genes. RNA-based drugs, including short interfering RNAs and antisense oligonucleotides, are particularly promising examples of this newer class of biologics. For over two decades, researchers have been trying to overcome major challenges for utilizing such RNAs in a therapeutic context, including intracellular delivery, stability, and immune response activation. This research is finally beginning to bear fruit as the first RNA drugs gain FDA approval and more advance to the final phases of clinical trials. Furthermore, the recent advent of CRISPR, an RNA-guided gene-editing technology, as well as new strides in the delivery of messenger RNA transcribed in vitro, have triggered a major expansion of the RNA-therapeutics field. In this review, we discuss the challenges for clinical translation of RNA-based therapeutics, with an emphasis on recent advances in delivery technologies, and present an overview of the applications of RNA-based drugs for modulation of gene/protein expression and genome editing that are currently being investigated both in the laboratory as well as in the clinic.
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