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

Cell, Год журнала: 2023, Номер 186(18), С. 3983 - 4002.e26

Опубликована: Авг. 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.

Язык: Английский

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CRISPR technologies for genome, epigenome and transcriptome editing

Nature Reviews Molecular Cell Biology, Год журнала: 2024, Номер 25(6), С. 464 - 487

Опубликована: Фев. 2, 2024

Язык: Английский

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Targeted genome-modification tools and their advanced applications in crop breeding

Nature Reviews Genetics, Год журнала: 2024, Номер 25(9), С. 603 - 622

Опубликована: Апрель 24, 2024

Язык: Английский

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Chromatin context-dependent regulation and epigenetic manipulation of prime editing

Cell, Год журнала: 2024, Номер 187(10), С. 2411 - 2427.e25

Опубликована: Апрель 11, 2024

We set out to exhaustively characterize the impact of cis-chromatin environment on prime editing, a precise genome engineering tool. Using highly sensitive method for mapping genomic locations randomly integrated reporters, we discover massive position effects, exemplified by editing efficiencies ranging from ∼0% 94% an identical target site and edit. Position effects efficiency are well predicted chromatin marks, e.g., positively H3K79me2 negatively H3K9me3. Next, developed multiplex perturbational framework assess interaction trans-acting factors with outcomes. Applying this DNA repair factors, identify HLTF as context-dependent repressor editing. Finally, several lines evidence suggest that active transcriptional elongation enhances Consistent this, show can robustly decrease or increase preceding it CRISPR-mediated silencing activation, respectively.

Язык: Английский

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High-throughput evaluation of genetic variants with prime editing sensor libraries

Nature Biotechnology, Год журнала: 2024, Номер unknown

Опубликована: Март 12, 2024

Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install evaluate genetic variants, but previous approaches have limited by the variable efficiency prime guide RNAs. Here we present high-throughput sensor strategy couples RNAs with synthetic versions their cognate target sites quantitatively assess functional impact endogenous variants. We screen over 1,000 cancer-associated variants TP53-the most frequently mutated gene in cancer-to identify alleles p53 function mechanistically diverse ways. find certain TP53 particularly those oligomerization domain, display opposite phenotypes exogenous overexpression systems. Our results emphasize physiological importance dosage shaping native stoichiometry protein-protein interactions, establish framework for studying sequence context at scale.

Язык: Английский

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Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells

Cell Reports Medicine, Год журнала: 2024, Номер 5(5), С. 101544 - 101544

Опубликована: Май 1, 2024

Prime editing is a recent, CRISPR-derived genome technology capable of introducing precise nucleotide substitutions, insertions, and deletions. Here, we present prime approaches to correct L227R- N1303K-CFTR, two mutations that cause cystic fibrosis are not eligible for current market-approved modulator therapies. We show that, upon DNA correction the CFTR gene, complex glycosylation, localization, and, most importantly, function protein restored in HEK293T 16HBE cell lines. These findings were subsequently validated patient-derived rectal organoids human nasal epithelial cells. Through analysis predicted experimentally identified candidate off-target sites primary stem cells, confirm previous reports on high editor (PE) specificity its potential curative CF gene therapy. To facilitate future screening genetic strategies translational model, machine learning algorithm was developed dynamic quantification (DETECTOR: "detection targeted organoids").

Язык: 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, Год журнала: 2024, Номер unknown

Опубликована: Июль 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

Язык: Английский

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CRISPR–Cas applications in agriculture and plant research

Nature Reviews Molecular Cell Biology, Год журнала: 2025, Номер unknown

Опубликована: Март 7, 2025

Язык: Английский

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High-throughput PRIME-editing screens identify functional DNA variants in the human genome

Molecular Cell, Год журнала: 2023, Номер 83(24), С. 4633 - 4645.e9

Опубликована: Дек. 1, 2023

Despite tremendous progress in detecting DNA variants associated with human disease, interpreting their functional impact a high-throughput and single-base resolution manner remains challenging. Here, we develop pooled prime-editing screen method, PRIME, that can be applied to characterize thousands of coding non-coding single experiment high reproducibility. To showcase its applications, first identified essential nucleotides for 716 bp MYC enhancer via PRIME-mediated analysis. Next, PRIME functionally 1,304 genome-wide association study (GWAS)-identified breast cancer 3,699 from ClinVar. We discovered 103 156 uncertain significance are affecting cell fitness. Collectively, demonstrate is capable characterizing genetic at scale, advancing accurate genome annotation disease risk prediction, diagnosis, therapeutic target identification.

Язык: Английский

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Machine learning prediction of prime editing efficiency across diverse chromatin contexts

Nature Biotechnology, Год журнала: 2024, Номер unknown

Опубликована: Июнь 21, 2024

Язык: Английский

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