Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency

Article Biochemical Research Methods

Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag

Shun-Qing Liang et al.

Summary: PE-tag is a genome-wide approach for identifying potential off-target sites of prime editors, enabling high-throughput profiling and safety evaluation.

NATURE METHODS (2023)

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Article Biotechnology & Applied Microbiology

A flexible split prime editor using truncated reverse transcriptase improves dual-AAV delivery in mouse liver

Chunwei Zheng et al.

Summary: Prime editor (PE) has great potential for gene therapy, but it is challenging to deliver PE in vivo. In this study, a compact PE without the RNase H domain was developed, which showed comparable editing efficiency with full-length PE. Using a Cas9 split site, robust editing was achieved in both cells and mouse liver. Furthermore, the compact PE efficiently mediated gene insertion in mouse liver without the stop codon read-through effect observed with full-length PE, indicating its potential for advancing prime editing in vivo.

MOLECULAR THERAPY (2022)

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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

Precise genomic deletions using paired prime editing

Junhong Choi et al.

Summary: The PRIME-Del method induces deletions with high precision using a pair of prime editing sgRNAs, outperforming CRISPR-Cas9 and sgRNA pairs in programming deletions up to 10 kb. This method can be broadly useful for precise, flexible programming of genomic deletions and potentially other rearrangements.

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 Multidisciplinary Sciences

Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair

J. Ferreira da Silva et al.

Summary: Prime editing is a powerful genome engineering approach that can introduce various modifications into the genome. This study identifies the mismatch repair pathway as inhibitory for Prime editing, and loss of mismatch repair enhances its efficiency.

NATURE COMMUNICATIONS (2022)

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Article Biotechnology & Applied Microbiology

CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells

Karl Petri et al.

Summary: This study demonstrated prime editing using purified ribonucleoprotein complexes, achieving desired edits in zebrafish embryos and human cells. However, unintended insertions, deletions, and guide RNA scaffold incorporations were also observed.

NATURE BIOTECHNOLOGY (2022)

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Article Biotechnology & Applied Microbiology

Dual-AAV delivering split prime editor system for in vivo genome editing

Shengyao Zhi et al.

Summary: Prime editor (PE) is a new genome editing tool that has the potential to correct the majority of known human genetic disease-related mutations. In this study, split-PEs were constructed and delivered using dual adenoassociated viruses (AAVs), successfully mediating gene editing in human cells and adult mouse retina.

MOLECULAR THERAPY (2022)

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Article Biotechnology & Applied Microbiology

A split prime editor with untethered reverse transcriptase and circular RNA template

Bin Liu et al.

Summary: This study reports a split prime editor (sPE) that can efficiently perform genome editing and shows potential therapeutic effects in a mouse model. Compared to previous split prime editors, this system reduces the complexity of the constructs and improves efficiency.

NATURE BIOTECHNOLOGY (2022)

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Article Biochemical Research Methods

Efficient targeted insertion of large DNA fragments without DNA donors

Jinlin Wang et al.

Summary: GRAND editing is a technique that enables efficient insertion of large DNA fragments without the need for DNA donors. By using a pair of prime editing guide RNAs and nonhomologous reverse transcription templates, it achieves high insertion efficiency in multiple cell lines and non-dividing cells.

NATURE METHODS (2022)

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Article Multidisciplinary Sciences

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

Xiaosa Li et al.

Summary: In this study, the authors optimized the gene editing system to increase the efficiency of Prime editor by altering the secondary structure of the guide RNA and introducing mutations. The developed strategies achieved highly efficient Prime editing at previously uneditable sites.

NATURE COMMUNICATIONS (2022)

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Article Biotechnology & Applied Microbiology

A truncated reverse transcriptase enhances prime editing by split AAV vectors

Zongliang Gao et al.

Summary: Prime editing is a novel genome editing technology that allows for more efficient and precise editing of the genome. This study made advancements in improving editing efficiencies by optimizing the reverse transcriptase moiety and addressing limitations of viral vector size. These improvements resulted in a codon-optimized and size-minimized prime editor that showed superior editing efficiency in in vivo applications.

MOLECULAR THERAPY (2022)

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Article Biology

Highly efficient generation of isogenic pluripotent stem cell models using prime editing

Hanqin Li et al.

Summary: Recent development of prime editing technology has the potential to simplify the generation of human pluripotent stem cell-based disease models. It is more efficient and precise than conventional gene editing methods, particularly in generating disease-associated mutations. By optimizing the delivery modalities, editing efficiency can be further improved.

ELIFE (2022)

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Article Genetics & Heredity

Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors

Sukanya Iyer et al.

Summary: This study describes the use of single-stranded DNA (ssDNA) phage to efficiently produce long circular ssDNA (cssDNA) donors, which can serve as efficient and cost-effective templates for homology-directed repair (HDR) in mammalian cells. The cssDNA donors outperform linear ssDNA (lssDNA) donors in terms of integration frequency and template-driven repair efficiency. The study also develops a traffic light reporter (TLR) system for evaluating the efficiencies of different Cas9 or Cas12a nucleases in editing and HDR, and extends the analysis to evaluate fluorescent tag knockins at endogenous sites in HEK293T and K562 cells.

CRISPR JOURNAL (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 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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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 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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Letter Cell Biology

Enhancing prime editing by Csy4-mediated processing of pegRNA

Yao Liu et al.

CELL RESEARCH (2021)

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Article Biochemistry & Molecular Biology

Optimized nickase- and nuclease-based prime editing in human and mouse cells

Fatwa Adikusuma et al.

Summary: Precise genomic modification using prime editing (PE) has great potential for research and clinical applications, with high efficiency observed in HEK293T cells but lower efficiency in K562 and HeLa cells. To improve efficiency in the latter, researchers developed a nuclease prime editor which greatly increased editing initiation but also led to extra insertions during the editing process.

NUCLEIC ACIDS RESEARCH (2021)

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Article Multidisciplinary Sciences

Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain

Myungjae Song et al.

Summary: By fusing a Rad51 DNA-binding domain, researchers have created hyPE2 with improved editing efficiency compared to PE2.

NATURE COMMUNICATIONS (2021)

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Article Biotechnology & Applied Microbiology

Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor

Soo-Ji Park et al.

Summary: Prime editors, a novel genome-editing tool, can induce targeted mutations with high efficiency. By utilizing two strategies to improve editing efficiency, we successfully generated Igf2 mutant mice with editing frequencies of up to 47% and observed important phenotypic changes.

GENOME BIOLOGY (2021)

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Article Genetics & Heredity