Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure

Article Biotechnology & Applied Microbiology

Engineered pegRNAs improve prime editing efficiency

James W. Nelson et al.

Summary: Prime editing technology enhances editing efficiency by optimizing the structure of pegRNAs, preventing degradation of the 3' region from affecting system activity, without increasing the risk of off-target editing.

NATURE BIOTECHNOLOGY (2022)

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Article Biotechnology & Applied Microbiology

Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing

Andrew Anzalone et al.

Summary: The twin prime editing (twinPE) method described in this study allows for programmable replacement or excision of DNA sequences at endogenous human genomic sites without the need for double-strand DNA breaks (DSBs). When combined with a site-specific serine recombinase, twinPE enables targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and may synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.

NATURE BIOTECHNOLOGY (2022)

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Article Genetics & Heredity

Genomic and Transcriptomic Analyses of Prime Editing Guide RNA-Independent Off-Target Effects by Prime Editors

Runze Gao et al.

Summary: A sensitive platform was developed to profile guide-independent OT effects of Prime editors in human cells, showing undetectable guide RNA-independent OT effects of PE3, suggesting high editing specificity of its reverse-transcriptase moiety.

CRISPR JOURNAL (2022)

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Article Plant Sciences

A design optimized prime editor with expanded scope and capability in plants

Wen Xu et al.

Summary: This study demonstrated that using N-terminal reverse transcriptase-Cas9 nickase fusion and introducing multiple-nucleotide substitutions in the reverse transcriptase template can significantly enhance the efficiency of prime editing in plants and human cells. The optimized approach achieved higher editing frequencies compared to the original editor, Prime Editor 3, showing the potential to make previously non-editable target sites editable and expand the capabilities of prime editing in the future.

NATURE PLANTS (2022)

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Article Biotechnology & Applied Microbiology

Predicting the efficiency of prime editing guide RNAs in human cells

Hui Kwon Kim et al.

Summary: This study identified factors affecting PE2 efficiency through high-throughput evaluation and developed three computational models to predict pegRNA efficiency, which can be applied to edits of various types and positions. Spearman's correlations between 0.47 and 0.81 were found when testing the accuracy of the predictions using independent test data sets.

NATURE BIOTECHNOLOGY (2021)

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Article Engineering, Biomedical

A web tool for the design of prime-editing guide RNAs

Ryan D. Chow et al.

Summary: Prime editing allows diverse genomic alterations to be made at target sites without double-strand breaks or donor templates. However, designing pegRNAs can be complex and time-consuming, as they require customization for each edit. The web tool pegFinder facilitates the rapid design of pegRNAs for downstream experimentation, with the incorporation of sgRNA predictions and nomination of secondary nicking sgRNAs for improved editing efficiency.

NATURE BIOMEDICAL ENGINEERING (2021)

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Article Biochemistry & Molecular Biology

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

Peter J. Chen et al.

Summary: The study found that DNA mismatch repair (MMR) hinders prime editing and promotes undesired byproducts, but the efficiency of substitution, small insertion, and small deletion edits can be enhanced by developing PE4 and PE5 prime editing systems. Strategic silent mutations near the intended edit and optimization of prime editor protein also contribute to improved editing outcomes.

CELL (2021)

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Review Biotechnology & Applied Microbiology

Next-Generation CRISPR Technologies and Their Applications in Gene and Cell Therapy

C. M. Alejandra Zeballos et al.

Summary: The emergence of CRISPR nucleases has revolutionized biotechnology by providing an easy, efficient, and versatile platform for editing DNA. New CRISPR-based modalities have been developed to catalyze editing without the requirement for DNA double-strand break repair, offering promising therapeutic applications and facing challenges for clinical implementation.

TRENDS IN BIOTECHNOLOGY (2021)

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Article Multidisciplinary Sciences

PrimeDesign software for rapid and simplified design of prime editing guide RNAs

Jonathan Y. Hsu et al.

Summary: Prime editing guide RNA design is more complex than for standard CRISPR-based nucleases or base editors. Here the authors present PrimeDesign and PrimeVar for the rapid and simplified design of pegRNA and ngRNA combinations. PrimeVar is a comprehensive and searchable database that includes candidate pegRNA and ngRNA combinations for installing or correcting >68,500 pathogenic human genetic variants.

NATURE COMMUNICATIONS (2021)

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Article Biotechnology & Applied Microbiology

High-efficiency prime editing with optimized, paired pegRNAs in plants

Qiupeng Lin et al.

Summary: Designing prime binding sites with a certain melting temperature and using two pegRNAs can substantially enhance the efficiency of prime editing.

NATURE BIOTECHNOLOGY (2021)

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Article Biotechnology & Applied Microbiology

Genome-wide specificity of prime editors in plants

Shuai Jin et al.

Summary: The study systematically evaluated the mismatch tolerance, off-target editing, and genome-wide variants induced by prime editors in plant cells, showing high precision and low off-target effects in plant genome editing.

NATURE BIOTECHNOLOGY (2021)

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Article Cell Biology

Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations

Lijie Wang et al.

Summary: The study introduces a novel base editor system, tBE, which efficiently performs C-to-T editing by fusing a cleavable dCDI domain, reducing off-target mutations. The tBE system remains inactive at off-target sites, preventing unintended mutations, and activates for precise base editing at on-target sites.

NATURE CELL BIOLOGY (2021)

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Article Multidisciplinary Sciences

Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

Pengpeng Liu et al.

Summary: The study introduces an NLS-optimized SpCas9-based prime editor that enhances genome editing efficiency and demonstrates the potential to induce tumor formation and correct pathogenic mutations in adult mice through somatic cell editing. The use of dual adeno-associated virus (AAVs) for delivery of a split-intein prime editor further establishes the capability of this system for in vivo installation of sequence modifications with important implications for disease modeling and correction.

NATURE COMMUNICATIONS (2021)

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Article Biotechnology & Applied Microbiology