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

Liang, Shun-Qing; P, Liu; Ponnienselvan, Karthikeyan; Suresh, Sneha; Chen, Zexiang; Kramme, Christian; Chatterjee, Pranam; Zhu, Lihua Julie; Sontheimer, Erik J.; Xue, Wen; Wolfe, Scot A.

doi:10.1038/s41592-023-01859-2

Cited by 24 publications

(8 citation statements)

References 43 publications

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“…Importantly, Pah enu2 correction rates were sufficient to reduce Phe levels below 600 µmol/l, which is considered therapeutic for adult PKU patients. In line with the high specificity of prime editing described in previous studies 48 , we did not detect editing at experimentally validated off-target binding sites with both pegRNAs. Furthermore, hepatotropism of LNPs and AAV2/9 ensured that editing was largely restricted to hepatocytes.…”

Section: Discussionsupporting

confidence: 89%

Treatment of a genetic liver disease in mice through transient prime editor expression

Rothgangl,

Ioannidi,

Weber

et al. 2024

Preprint

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Prime editing is a versatile genome editing technology that does not rely on DNA double-strand break formation and homology-directed repair (HDR). This makes it a promising tool for correcting pathogenic mutations in tissues consisting predominantly of postmitotic cells, such as the liver. While recent studies have already demonstrated proof-of-concept for in vivo prime editing, the use of viral delivery vectors resulted in prolonged prime editor (PE) expression, posing challenges for clinical application. Here, we developed an in vivo prime editing approach where we delivered the pegRNA using self-complementary adeno-associated viral (scAAV) vectors and the prime editor using nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs). This methodology led to transient expression of the PE for 48h and 26% editing at the Dnmt1 locus using AAV doses of 2.5x1013 vector genomes (vg)/kg and a single dose of 3mg/kg mRNA-LNP. When targeting the pathogenic mutation in the Pahenu2 mouse model of phenylketonuria (PKU), we achieved 4.3% gene correction using an AAV dose of 2.5x1013 vg/kg and three doses of 2 mg/kg mRNA-LNP. Editing was specific to the liver and the intended locus, and was sufficient to reduce blood L-phenylalanine (Phe) levels from over 1500 umol/l to below the therapeutic threshold of 600 umol/l. Our study demonstrates the feasibility of in vivo gene correction in the liver with transient PE expression, bringing prime editing closer to clinical application.

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Section: Discussionsupporting

confidence: 89%

Treatment of a genetic liver disease in mice through transient prime editor expression

Rothgangl,

Ioannidi,

Weber

et al. 2024

Preprint

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“…3C , Left) ( 81 ). The recent prime editing technology has been shown to achieve a target specificity that is relatively superior to that of inducing indels by DNA cleavage with the existing CRISPR system ( 82 , 83 ). However, the disadvantage of the current prime editing technology is that, in vivo , the overall gene editing efficiency is low for various gene sites, and byproduct indels are generated when an advanced PE3 system is used to overcome low efficiency ( Fig.…”

Section: Methods To Overcome Off-target Effects In the Crispr Systemmentioning

confidence: 99%

Recent advances in genome engineering by CRISPR technology

Lee,

Oh,

Lee

2024

BMB Rep

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Due to the development of CRISPR technology, the era of effective editing of target genes has arrived. However, the off-target problem that occurs when recognizing target DNA due to the inherent nature of CRISPR components remains the biggest task to be overcome in the future. In this review, the principle of inducing such unintended off-target editing is analyzed from the structural aspect of CRISPR, and the methodology that has been developed to reduce off-target editing until now is summarized.

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“…Prime editing is a next generation genome engineering approach that allows DNA donor-free installation of various types of small genomic edits including insertions, deletions, and substitutions (Chen and Liu, 2023). Compared to more traditional approaches for precise genome editing such as homology-directed repair (HDR), prime editing induces comparably lower levels of undesired editing outcomes and off-targets (Anzalone et al, 2019, Liang et al, 2023). A typical prime editor (PE) comprises a Cas9 nickase fused to a reverse transcriptase (RT) via a flexible linker (Anzalone et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Improved nuclease-based prime editing by DNA repair modulation and pegRNA engineering

Antoniou,

Dacquay,

Selfjord

et al. 2024

Preprint

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Prime editing is a genome engineering tool that allows installation of small edits with high precision. However, prime editing efficiency and purity can vary widely across different edits, genomic targets, and cell types. Prime editing nuclease (PEn) utilizes a fully active Cas9 instead of the nickase employed in conventional prime editors. PEn is capable of editing sites resistant to nickase-based prime editors but induces more undesired editing events. In this work, we introduce two strategies to enhance PEn precision and efficiency. First, we apply a small molecule approach, selectively modulating DNA repair pathways, to improve PEn precision up to 9.8-fold and reduce off-target editing by 90%. Second, through pegRNA engineering, we devise a strategy that mitigates unintended pegRNA scaffold integration, which is a common prime editing by-product, enhancing precision up to 3.5-fold. We apply this approach to a specific type of PEn editing mediated through non-homologous end joining and use it to achieve efficient and precise prime editing in multiple human cell lines, primary human hepatocytes, and mouse embryos. Together, this work presents two general strategies to improve prime editing, overcomes the limitations of current PEn editors, and provides reliable and precise genome editing outcomes, a pivotal requirement for therapeutic applications.

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

Cited by 24 publications

References 43 publications

Treatment of a genetic liver disease in mice through transient prime editor expression

Treatment of a genetic liver disease in mice through transient prime editor expression

Recent advances in genome engineering by CRISPR technology

Improved nuclease-based prime editing by DNA repair modulation and pegRNA engineering

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