Prime editing for precise and highly versatile genome manipulation

Nature Reviews Genetics, Journal Year: 2022, Volume and Issue: 24(3), P. 161 - 177

Published: Nov. 7, 2022

Language: Английский

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Therapeutic in vivo delivery of gene editing agents

Cell, Journal Year: 2022, Volume and Issue: 185(15), P. 2806 - 2827

Published: July 1, 2022

In vivo gene editing therapies offer the potential to treat root causes of many genetic diseases. Realizing promise therapeutic in requires ability safely and efficiently deliver agents relevant organs tissues vivo. Here, we review current delivery technologies that have been used enable editing, including viral vectors, lipid nanoparticles, virus-like particles. Since no single modality is likely be appropriate for every possible application, compare benefits drawbacks each method highlight opportunities future improvements.

Language: Английский

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Phage-assisted evolution and protein engineering yield compact, efficient prime editors

Cell, Journal Year: 2023, Volume and Issue: 186(18), P. 3983 - 4002.e26

Published: Aug. 1, 2023

Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size improved efficiency. Using phage-assisted evolution, efficiencies compact reverse transcriptases by up 22-fold generated that are 516–810 base pairs smaller than the current-generation editor PEmax. We discovered different specialize types used this insight outperform PEmax PEmaxΔRNaseH, truncated dual-AAV delivery systems. Finally, Cas9 domains improve editing. These resulting (PE6a-g) enhance therapeutically relevant patient-derived fibroblasts primary human T-cells. PE6 variants also enable longer insertions be installed vivo following delivery, achieving 40% loxP insertion cortex murine brain, 24-fold improvement compared previous state-of-the-art editors.

Language: Английский

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In vivo somatic cell base editing and prime editing

Molecular Therapy, Journal Year: 2021, Volume and Issue: 29(11), P. 3107 - 3124

Published: Sept. 10, 2021

Recent advances in genome editing technologies have magnified the prospect of single-dose cures for many genetic diseases. For most disorders, precise DNA correction is anticipated to best treat patients. To install desired changes with high precision, our laboratory developed base editors (BEs), which can correct four common single-base substitutions, and prime editors, any substitution, insertion, and/or deletion over a stretch dozens pairs. Compared nuclease-dependent approaches that involve double-strand breaks (DSBs) often result large percentage uncontrolled outcomes, such as mixtures insertions deletions (indels), larger deletions, chromosomal rearrangements, offer greater efficiency fewer byproducts slowly dividing or non-dividing cells, those make up cells adult animals. Both viral non-viral vivo delivery methods now been used deliver animal models, establishing serve effective agents therapeutic This review summarizes examples somatic cell (post-natal) prospects future development.

Language: Английский

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Efficient prime editing in mouse brain, liver and heart with dual AAVs

Nature Biotechnology, Journal Year: 2023, Volume and Issue: 42(2), P. 253 - 264

Published: May 4, 2023

Abstract Realizing the promise of prime editing for study and treatment genetic disorders requires efficient methods delivering editors (PEs) in vivo. Here we describe identification bottlenecks limiting adeno-associated virus (AAV)-mediated vivo development AAV-PE vectors with increased PE expression, guide RNA stability modulation DNA repair. The resulting dual-AAV systems, v1em v3em PE-AAV, enable therapeutically relevant mouse brain (up to 42% efficiency cortex), liver 46%) heart 11%). We apply these systems install putative protective mutations Alzheimer’s disease astrocytes coronary artery hepatocytes. In PE-AAV caused no detectable off-target effects or significant changes enzymes histology. Optimized support highest unenriched levels reported date, facilitating potential diseases a component.

Language: Английский

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Precise genomic editing of pathogenic mutations in RBM20 rescues dilated cardiomyopathy

Science Translational Medicine, Journal Year: 2022, Volume and Issue: 14(672)

Published: Nov. 23, 2022

Mutations in RNA binding motif protein 20 ( RBM20 ) are a common cause of familial dilated cardiomyopathy (DCM). Many mutations cluster within an arginine/serine-rich (RS-rich) domain, which mediates nuclear localization. These induce mis-localization to form aberrant ribonucleoprotein (RNP) granules the cytoplasm cardiomyocytes and abnormal alternative splicing cardiac genes, contributing DCM. We used adenine base editing (ABE) prime (PE) correct pathogenic p.R634Q p.R636S RS-rich domain human isogenic induced pluripotent stem cell (iPSC)–derived cardiomyocytes. Using ABE R634Q iPSCs, we achieved 92% efficiency A-to-G editing, normalized restored localization RBM20, eliminated RNP granule formation. In addition, developed PE strategy R636S mutation iPSCs observed A-to-C at 40% efficiency. To evaluate potential for DCM treatment, also created Rbm20 R636Q mutant mice. Homozygous (R636Q/R636Q) mice severe dysfunction, heart failure, premature death. Systemic delivery components containing ABEmax-VRQR-SpCas9 single-guide by adeno-associated virus serotype 9 these function as assessed echocardiography extended life span. As seen sequencing analysis, correction rescued transcriptional profile treated R636Q/R636Q mice, compared gene expression untreated findings demonstrate precise genetic promising therapeutic approach

Language: Английский

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Prime editing: advances and therapeutic applications

Trends in biotechnology, Journal Year: 2023, Volume and Issue: 41(8), P. 1000 - 1012

Published: March 30, 2023

Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR–Cas)-mediated genome editing has revolutionized biomedical research and will likely change the therapeutic diagnostic landscape. However, CRISPR–Cas9, which edits DNA by activating double-strand break (DSB) repair pathways, is not always sufficient for gene therapy applications where precise mutation required. Prime editing, latest revolution in genome-editing technologies, can achieve any possible base substitution, insertion, or deletion without requirement DSBs. prime still its infancy, further development needed to improve efficiency delivery strategies applications. We summarize developments optimization of editor (PE) variants with improved precision. Moreover, we highlight some potential

Language: Английский

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Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo

Nature Biotechnology, Journal Year: 2024, Volume and Issue: 42(10), P. 1526 - 1537

Published: Jan. 8, 2024

Prime editing enables precise installation of genomic substitutions, insertions and deletions in living systems. Efficient vitro vivo delivery prime components, however, remains a challenge. Here we report editor engineered virus-like particles (PE-eVLPs) that deliver proteins, guide RNAs nicking single as transient ribonucleoprotein complexes. We systematically v3 v3b PE-eVLPs with 65- to 170-fold higher efficiency human cells compared PE-eVLP construct based on our previously reported base eVLP architecture. In two mouse models genetic blindness, injections resulted therapeutically relevant levels the retina, protein expression restoration partial visual function rescue. Optimized support ribonucleoproteins, enhancing potential safety by reducing off-target obviating possibility oncogenic transgene integration.

Language: Английский

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Designing and executing prime editing experiments in mammalian cells

Nature Protocols, Journal Year: 2022, Volume and Issue: 17(11), P. 2431 - 2468

Published: Aug. 8, 2022

Language: Английский

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Predicting prime editing efficiency and product purity by deep learning

Nature Biotechnology, Journal Year: 2023, Volume and Issue: 41(8), P. 1151 - 1159

Published: Jan. 16, 2023

Language: Английский

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