DNA base editing in nuclear and organellar genomes

Tan, Junjie; Forner, Joachim; Karcher, Daniel; Bock, Ralph

doi:10.1016/j.tig.2022.06.015

Cited by 23 publications

(6 citation statements)

References 192 publications

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“…CRISPR technology is an efficient and simple method for introducing specific edit to the genome. [ 1,2 ] However, the efficiency of desired edits is very low in traditional CRISPR/Cas9 HDR, resulting in the difficulty to generate successful knock‐in cells. Prime editing enables a more flexible way for genome editing.…”

Section: Discussionmentioning

confidence: 99%

“…The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system has revolutionized the field of genome editing. [ 1,2 ] Prime editing (PE) is a cutting‐edge genome editing technique derived from the CRISPR/Cas system. [ 3 ] It enables highly precise, targeted modifications to the genome without creating DNA double‐strand breaks (DSB).…”

Section: Introductionmentioning

confidence: 99%

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A refined Uni‐vector prime editing system improves genome editing outcomes in mammalian cells

Huang,

Chiu,

Chou

et al. 2024

Biotechnology Journal

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Prime editing is an advanced technology in CRISPR/Cas research with increasing numbers of improved methodologies. The original multi‐vector method hampers the efficiency and precision of prime editing and also has inherent difficulty in generating homozygous mutations in mammalian cells. To overcome these technical issues, we developed a Uni‐vector prime editing system, wherein the major components for prime editing were constructed in all‐in‐one plasmids, pPE3‐pPuro and pePEmax‐pPuro. The Uni‐vector prime editing plasmids enhance the editing efficiency of prime editing and improved the generation of homozygous mutated mammalian cell lines. The editing efficiency is dependent of the transfection efficiency. Remarkably, the Uni‐vector ePE5max system achieved an impressive editing rate approximately 79% in average, even in cell lines that are traditionally difficult to transfect, such as FaDu cell line. Furthermore, it resulted in a high frequency of homozygous knocked‐in cells, with a rate of 99% in HeLa and 85% in FaDu cells. Together, our Uni‐vector approach simplifies the delivery of editing components and improves the editing efficiency, especially in cells with low transfection efficiency. This approach presents an advancement in the field of prime editing.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A refined Uni‐vector prime editing system improves genome editing outcomes in mammalian cells

Huang,

Chiu,

Chou

et al. 2024

Biotechnology Journal

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“…Many BEs generate a wide array of mutations and MFs in cell populations: on‐targets with the highest, followed by recurring (often predictable) off‐targets, and random (unpredictable) off‐targets with the lowest (reviewed in, Jeong et al, 2020; Tan et al, 2022). Mutations in this last group are ultrarare and spread across the genomes of the cell population, which makes their MFs undetectable by traditional sequencing.…”

Section: Discussionmentioning

confidence: 99%

Unbiased whole genome detection of ultrarare off‐target mutations in genome‐edited cell populations by HiFi sequencing

Miranda

Fenner

McKinzie

et al. 2023

Environ and Mol Mutagen

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DNA base editors (BEs) composed of a nuclease‐deficient Cas9 fused to a DNA‐modifying enzyme can achieve on‐target mutagenesis without creating double‐strand DNA breaks (DSBs). As a result, BEs generate far less DNA damage than traditional nuclease‐proficient Cas9 systems, which do rely on the creation of DSBs to achieve on‐target mutagenesis. The inability of BEs to create DSBs makes the detection of their undesired off‐target effects very difficult. PacBio HiFi sequencing can efficiently detect ultrarare mutations resulting from chemical mutagenesis in whole genomes with a sensitivity ~1 × 10−8 mutations per base pair. In this proof‐of‐principle study, we evaluated whether this technique could also detect the on‐ and off‐target mutations generated by a cytosine‐to‐thymine (C>T) BE targeting the LacZ gene in Escherichia coli (E. coli). HiFi sequencing detected on‐target mutant allele fractions ranging from ~7% to ~63%, depending on the single‐guide RNA (sgRNA) used, while no on‐target mutations were detected in controls lacking the BE. The presence of the BE resulted in a ~3‐fold increase in mutation frequencies compared to controls lacking the BE, irrespective of the sgRNA used. These increases were mostly composed of C:G>T:A substitutions distributed throughout the genome. Our results demonstrate that HiFi sequencing can efficiently identify on‐ and off‐target mutations in cell populations that have undergone genome editing.

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“…CBEs carry a cytosine deaminase and cause C-to-T on the target sequence. In ABEs, the combination of adenosine deaminase and Cas9 protein induces the conversion of A-to-G transition by catalyzing the oxidative deamination of deoxyadenosine to deoxyinosine [6]. Combining transcriptional repressor or activator domains with deactivated Cas9 (dCas9), which has both cleavage domains, RuvC and HNH domains, inactivated, enables CRISPR-mediated transcriptional activation [7].…”

Section: Crispr-cas9 Systemmentioning

confidence: 99%

Applications of the CRISPR-Cas9 system in cancer models

Yang

2023

TNS

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Cancer has a high mortality and prevalence rate in the world. CRISPR-Cas9 is one of the novel and most common gene-editing techniques. Compared with the first two generations of gene-editing technologies, CRISPR-Cas9 system has the advantages of easy design, low cost, high efficiency and so on. sgRNA guides Cas9 to the site of the targeted gene, and Cas9 cuts the DNA strand at that site, triggering the NHEJ or HDR mechanism so as to achieve the purpose of deletion or insertion. CRISPR-Cas9 can be combined with other factors for other purposes, such as CRISPRa, CRISPRi, and base editing. The CRISPR system now has been used extensively for research into biological mechanisms and disease treatments. Since cancer is controlled by genes, a number of researchers in recent years have looked at using the CRISPR system to treat cancer. The CRISPR technology has greatly improved our understanding of cancer and the factors that affect it, and has had a major impact on the study and treatment of cancer. CRISPR gene editing can quickly and efficiently generate gene knockouts and regulate gene expression to identify relevant genes that influence cancer growth. This review systematically introduces CRISPR-Cas9 and its application methods, delivery modes, and discusses some studies using cell lines and organoids in vitro and animal models for cancer therapy in vivo.

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DNA base editing in nuclear and organellar genomes

Cited by 23 publications

References 192 publications

A refined Uni‐vector prime editing system improves genome editing outcomes in mammalian cells

A refined Uni‐vector prime editing system improves genome editing outcomes in mammalian cells

Unbiased whole genome detection of ultrarare off‐target mutations in genome‐edited cell populations by HiFi sequencing

Applications of the CRISPR-Cas9 system in cancer models

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