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Research progress of gene correction for genetic diseases via in vivo prime editing.
Curative therapies for genetic diseases caused by pathogenic mutations demand precise and safe genome editing technologies. Prime editing (PE), a CRISPR-Cas9-derived technology that enables precise base conversions without double-strand breaks (DSBs), has emerged as a transformative tool with the ability to correct 89% of human pathogenic mutations. However, transitioning PE from preclinical models to clinical applications faces critical challenges, particularly in the development of in vivo delivery systems and scalable manufacturing. This review systematically summarizes advances of in vivo PE therapeutics, focusing on the therapeutic outcomes across some retinal, metabolic, and hematologic genetic diseases. We further dissect barriers to clinical translation, including in the immunogenicity of viral vectors, off-target risks in nondividing cells, and large-scale production bottlenecks. By highlighting the unique advantages of in vivo PE and proposing actionable solutions for scalable manufacturing, the work provides a roadmap for accelerating in vivo PE-based therapies toward clinical reality.
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CRISPys: Optimal sgRNA Design for Editing Multiple Members of a Gene Family Using the CRISPR System.
The development of the CRISPR-Cas9 system in recent years has made eukaryotic genome editing, and specifically gene knockout for reverse genetics, a simple and effective task. The system is directed to a genomic target site by a programmed single-guide RNA (sgRNA) that base-pairs with it, subsequently leading to site-specific modifications. However, many gene families in eukaryotic genomes exhibit partially overlapping functions, and thus, the knockout of one gene might be concealed by the function of the other. In such cases, the reduced specificity of the CRISPR-Cas9 system, which may lead to the modification of genomic sites that are not identical to the sgRNA, can be harnessed for the simultaneous knockout of multiple homologous genes. We introduce CRISPys, an algorithm for the optimal design of sgRNAs that would potentially target multiple members of a given gene family. CRISPys first clusters all the potential targets in the input sequences into a hierarchical tree structure that specifies the similarity among them. Then, sgRNAs are proposed in the internal nodes of the tree by embedding mismatches where needed, such that the efficiency to edit the induced targets is maximized. We suggest several approaches for designing the optimal individual sgRNA and an approach to compute the optimal set of sgRNAs for cases when the experimental platform allows for more than one. The latter may optionally account for the homologous relationships among gene-family members. We further show that CRISPys outperforms simpler alignment-based techniques by in silico examination over all gene families in the Solanum lycopersicum genome.
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Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells.
Base editors show promise for treating human genetic diseases, but most current systems use deaminases, which cause off-target effects and are limited in editing type. In this study, we constructed deaminase-free base editors for cytosine (DAF-CBE) and thymine (DAF-TBE), which contain only a cytosine-DNA or a thymine-DNA glycosylase (CDG/TDG) variant, respectively, tethered to a Cas9 nickase. Multiple rounds of mutagenesis by directed evolution in Escherichia coli generated two variants with enhanced base-converting activity-CDG-nCas9 and TDG-nCas9-with efficiencies of up to 58.7% for C-to-A and 54.3% for T-to-A. DAF-BEs achieve C-to-G/T-to-G editing in mammalian cells with minimal Cas9-dependent and Cas9-independent off-target effects as well as minimal RNA off-target effects. Additional engineering resulted in DAF-CBE2/DAF-TBE2, which exhibit altered editing windows from the 5' end to the middle of the protospacer and increased C-to-G/T-to-G editing efficiency of 3.5-fold and 1.2-fold, respectively. Compared to prime editing or CGBEs, DAF-BEs expand conversion types of base editors with similar efficiencies, smaller sizes and lower off-target effects.
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[Plant prime editing technique: a new genome editing tool for plants].
The CRISPR/Cas9 based prime editing (PE) technique enables all 12 types of base substitutions and precise small DNA deletions or insertions without generating DNA double-strand breaks. Prime editing has been successfully applied in plants and plays important roles in plant precision breeding. Although plant prime editing (PPE) can substantially expand the scope and capabilities of precise genome editing in plants, its editing efficiency still needs to be further improved. Here, we review the development of PPE technique, and introduce structural composition, advantages and limitations of PPE. Strategies to improve the PPE editing efficiency, including the Tm-directed PBS length design, the RT template length, the dual-pegRNA strategy, the PlantPegDesigner website, and the strategies for optimizing the target proteins of PPE, were highlighted. Finally, the prospects of future development and application of PPE were discussed.
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Precise Correction of Heterozygous SHOX2 Mutations in hiPSCs Derived from Patients with Atrial Fibrillation via Genome Editing and Sib Selection.
Patient-specific human induced pluripotent stem cells (hiPSCs) offer unprecedented opportunities for the investigation of multigenic disease, personalized medicine, and stem cell therapy. For heterogeneous diseases such as atrial fibrillation (AF), however, precise correction of the associated mutation is crucial. Here, we generated and corrected hiPSC lines from two AF patients carrying different heterozygous SHOX2 mutations. We developed a strategy for the scarless correction of heterozygous mutations, based on stochastic enrichment by sib selection, followed by allele quantification via digital PCR and next-generation sequencing to detect isogenic subpopulations. This allowed enriching edited cells 8- to 20-fold. The method does not require antibiotic selection or cell sorting and can be easily combined with base-and-prime editing approaches. Our strategy helps to overcome low efficiencies of homology-dependent repair in hiPSCs and facilitates the generation of isogenic control lines that represent the gold standard for modeling complex diseases in vitro.
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CRISPR-Cas9 guided RNA-based model for the silencing of spinal bulbar muscular atrophy: A functional genetic disorder.
This study explores a novel therapeutic approach for spinal bulbar muscular atrophy (SBMA), a neurodegenerative disorder caused by a mutation in the Androgen Receptor (AR) gene. The aim is to investigate the potential of CRISPR-Cas9 technology in targeting the mutant AR gene to inhibit its production. The objectives include assessing the accuracy and efficacy of CRISPR-Cas9 guided RNAs in silencing the mutant gene and evaluating the feasibility of this approach as a treatment for SBMA. Computational and in-silico approaches are used to evaluate the feasibility of using CRISPR-Cas9 technology for treating SBMA. Computational analysis is used to design CRISPR-Cas9 guided RNAs targeting the mutant AR gene, assessing their on-target and off-target scores, GC content, and structural accuracy. In-silico simulations predict the potential therapeutic outcomes of the CRISPR-Cas9 approach in an artificial environment. Three guided RNA (gRNA) sequences were designed using the CHOPCHOP tool, targeting specific regions of the AR gene with high efficiency and 100% match. These gRNAs demonstrated effective targeting with minimal off-target scores and optimal GC content. Additionally, lentiCRISPR v2 plasmids were designed for the delivery of CRISPR materials, enabling high-efficiency multiplex genome editing of the AR gene. Thermodynamic ensemble predictions indicated favorable secondary structure stability of the designed gRNAs, further supporting their suitability for gene editing. The evaluation of designed gRNAs confirmed their strong binding ability to the target sequences, validating their potential as effective tools for genome editing. The study highlights the potential of CRISPR-Cas9 technology for targeting the Androgen Receptor gene associated with spinal bulbar muscular atrophy (SBMA). The findings support the feasibility of this approach for gene editing and suggest further exploration in preclinical and clinical settings. Recommendations include continued research to optimize CRISPR-Cas9 delivery methods and enhance specificity for therapeutic applications in SBMA.
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Robust Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis elegans.
CRISPR-based genome editing using ribonucleoprotein complexes and synthetic single-stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs-such as green fluorescent protein tags remains challenging in many systems. Here, we describe a streamlined and optimized editing protocol for the nematode Caenorhabditis elegans We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging 14 Argonaute proteins in C. elegans using ssODN donors. In addition, we describe a novel PCR-based, partially single-stranded, "hybrid" donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci, achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems, and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.
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Towards precise large genomic fragment deletion.
The prime editing (PE) system can install small insertions and deletions in addition to various base substitutions. Two recent studies published in Nature Biotechnology by Choi et al. and Jiang et al. report that the system can also be tweaked for efficient and precise deletions of large DNA fragments.
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Oligo-seq protocol for mapping DNA motifs targeted by base editors.
Determining which DNA sequences are preferentially targeted by base editors is critical for understanding how APOBECs, AID, and other CRISPR-Cas9 base editors edit DNA in cells or improve their editing efficiency. We have developed Oligo-seq, an in vitro sequencing-based method to identify the preferred sequence motifs targeted by these enzymes. This assay monitors DNA deaminase activity on DNA oligonucleotides containing random nucleotides and/or DNA structures and determines by sequencing which sequences are preferentially deaminated. For complete details on the use and execution of this protocol, please refer to Sanchez et al.1.
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CRISPR/Cas9 in zebrafish: An attractive model for FBN1 genetic defects in humans.
BACKGROUND: Mutations in the fibrillin-1 gene (FBN1) are associated with various heritable connective tissue disorders (HCTD). The most studied HCTD is Marfan syndrome. Ninety percent of Marfan syndrome is caused by mutations in the FBN1 gene. The zebrafish share high genetic similarity to humans, representing an ideal model for genetic research of human diseases. This study aimed to generate and characterize fbn1+/- mutant zebrafish using the CRISPR/Cas9 gene-editing technology. METHODS: CRISPR/Cas9 was applied to generate an fbn1 frameshift mutation (fbn1+/- ) in zebrafish. F1 fbn1+/- heterozygotes were crossed with transgenic fluorescent zebrafish to obtain F2 fbn1+/- zebrafish. Morphological abnormalities were assessed in F2 fbn1+/- zebrafish by comparing with the Tuebingen (TU) wild-type controls at different development stages. RESULTS: We successfully generated a transgenic line of fbn1+/- zebrafish. Compared with TU wild-type zebrafish, F2 fbn1+/- zebrafish exhibited noticeably decreased pigmentation, increased lengths, slender body shape, and abnormal cardiac blood flow from atrium to ventricle. CONCLUSION: We generated the first fbn1+/- zebrafish model using CRISPR/Cas9 gene-editing approach to mimic FBN1 genetic defects in humans, providing an attractive model of Marfan syndrome and a method to determine the pathogenicity of gene mutation sites.
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