CRISPR technologies for genome, epigenome and transcriptome editing

Nature Reviews Molecular Cell Biology, Journal Year: 2024, Volume and Issue: 25(6), P. 464 - 487

Published: Feb. 2, 2024

Language: Английский

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Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos

Nature Biotechnology, Journal Year: 2023, Volume and Issue: 42(4), P. 638 - 650

Published: June 15, 2023

Language: Английский

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Prediction of efficiencies for diverse prime editing systems in multiple cell types

Cell, Journal Year: 2023, Volume and Issue: 186(10), P. 2256 - 2272.e23

Published: April 28, 2023

Language: Английский

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CRISPR engineering in organoids for gene repair and disease modelling

Nature Reviews Bioengineering, Journal Year: 2023, Volume and Issue: 1(1), P. 32 - 45

Published: Jan. 19, 2023

Language: Английский

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In vivo HSC prime editing rescues Sickle Cell Disease in a mouse model

Blood, Journal Year: 2023, Volume and Issue: unknown

Published: Feb. 17, 2023

Sickle Cell Disease (SCD) is a monogenic disease caused by nucleotide mutation in the β-globin gene. Current gene therapy studies are mainly focused on lentivirus vector-mediated addition or CRISPR/Cas9-mediated fetal globin reactivation, leaving root cause unfixed. We developed vectorized prime editing system that can directly repair SCD hematopoietic stem cells (HSCs) vivo mouse model (CD46/Townes mice). Our approach involved single intravenous injection of non-integrating, editor-expressing virus vector into mobilized CD46/Townes mice and low-dose drug selection vivo. This procedure resulted correction ~40% bS alleles HSCs. On average 43% HbS was replaced HbA thereby greatly mitigating phenotypes. Transplantation secondary recipients demonstrated long-term repopulating HSCs were edited. Highly efficient target site achieved with minimal generation insertions deletions no detectable off-target editing. Because its simplicity portability, our has potential for application resource-poor countries where prevalent.

Language: Английский

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Dual-AAV split prime editor corrects the mutation and phenotype in mice with inherited retinal degeneration

Signal Transduction and Targeted Therapy, Journal Year: 2023, Volume and Issue: 8(1)

Published: Feb. 6, 2023

The prime editor (PE) can edit genomes with almost any intended changes, including all 12 possible types of base substitutions, small insertions and deletions, their combinations, without the requirement for double strand breaks or exogenous donor templates. PE demonstrates possibility correcting a variety disease-causing mutations might expand therapeutic application gene editing. In this study, was optimized based on dual-adeno-associated virus (AAV) split-intein system in vitro by screening different split sites inteins. We found that splitting before amino acid 1105(Ser) SpCas9 Rma intein resulted highest on-target orientations pegRNA nicking sgRNA AAV vector were further optimized. To test vivo performance dual-AAV split-PE3, it delivered subretinal injection rd12 mice inherited retinal disease Leber congenital amaurosis. editors corrected pathogenic mutation up to 16% efficiency precise way, no detectable off-target edits, restored RPE65 expression, rescued visual function, preserved photoceptors. Our findings establish framework preclinical development motivate testing treatment diseases caused various mutations.

Language: Английский

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Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells

Nature Biomedical Engineering, Journal Year: 2024, Volume and Issue: unknown

Published: July 10, 2024

Abstract Prime editing (PE) enables precise and versatile genome without requiring double-stranded DNA breaks. Here we describe the systematic optimization of PE systems to efficiently correct human cystic fibrosis (CF) transmembrane conductance regulator ( CFTR ) F508del, a three-nucleotide deletion that is predominant cause CF. By combining six efficiency optimizations for PE—engineered guide RNAs, PEmax architecture, transient expression dominant-negative mismatch repair protein, strategic silent edits, PE6 variants proximal ‘dead’ single-guide RNAs—we increased correction efficiencies F508del from less than 0.5% in HEK293T cells 58% immortalized bronchial epithelial (a 140-fold improvement) 25% patient-derived airway cells. The also resulted minimal off-target editing, edit-to-indel ratios 3.5-fold greater those achieved by nuclease-mediated homology-directed repair, functional restoration ion channels over 50% wild-type levels (similar via combination treatment with elexacaftor, tezacaftor ivacaftor) primary Our findings support feasibility durable one-time

Language: Английский

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Precise genome-editing in human diseases: mechanisms, strategies and applications

Signal Transduction and Targeted Therapy, Journal Year: 2024, Volume and Issue: 9(1)

Published: Feb. 26, 2024

Precise genome-editing platforms are versatile tools for generating specific, site-directed DNA insertions, deletions, and substitutions. The continuous enhancement of these has led to a revolution in the life sciences, which promises deliver novel therapies genetic disease. can be traced back 1950s with discovery DNA's double-helix and, after 70 years development, evolved from crude vitro applications wide range sophisticated capabilities, including vivo applications. Nonetheless, precise faces constraints such as modest efficiency, delivery challenges, off-target effects. In this review, we explore genome-editing, focus on introduction landmark events its history, various platforms, systems, First, discuss history genome-editing. Second, describe current state strategies explain how techniques offer unprecedented precision versatility modifying human genome. Third, introduce systems used deploy components through DNA, RNA, RNPs. Finally, summarize labeling endogenous genes, screening variants, molecular recording, disease models, gene therapy, ex therapy potential future advances.

Language: Английский

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Advanced Delivery Systems for Gene Editing: A Comprehensive Review from the GenE-HumDi COST Action Working group

Molecular Therapy — Nucleic Acids, Journal Year: 2025, Volume and Issue: 36(1), P. 102457 - 102457

Published: Jan. 18, 2025

Language: Английский

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Mismatch prime editing gRNA increased efficiency and reduced indels

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: Jan. 2, 2025

Prime editing enables precise and efficient genome editing, but its efficacy is hindered by pegRNA's 3' extension, forming secondary structures due to high complementarity with the protospacer. The continuous presence of prime system also leads unintended indel formation, raising safety concerns for therapeutic applications. To address these challenges, we develop a mismatched pegRNA (mpegRNA) strategy that introduces bases into protospacer, reducing structure preventing sustained activity. Our findings show mpegRNA enhances efficiency up 2.3 times reduces levels 76.5% without compromising performance. Combining epegRNA further increases 14-fold, or 2.4-fold in PE4max/PE5max systems, underscoring potential research therapy. AlphaFold 3 analysis suggests optimal contributes significantly improved outcomes. Overall, advances technology, improving while indels. extension. Here authors reduce formation can increase

Language: Английский

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