Generation of C-to-G transversion in mouse embryos via CG editors

Cao, Tianqi; Liu, Simiao; Qiu, Yanling; Gao, Min; Wu, Jinni; Wu, Guifang; Liang, Puping; Huang, Junjiu

doi:10.1007/s11248-022-00313-x

Cited by 5 publications

(1 citation statement)

References 45 publications

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“…The well-established BEs comprise a nickase Cas9 fused to a deaminase enzyme ( Rees and Liu, 2017 ) and exist in two types, the cytosine base editors (CBEs), which are composed of a cytidine deaminase and are able to convert a cytidine (C) into a thymidine (T) base ( Komor et al, 2016 ), and the adenine base editors (ABE), which are composed of an adenine deaminase and are able to convert an adenine (A) into a guanine (G) base ( Gaudelli et al, 2017 ). In a rapidly progressing expansion of our arsenal of editors, PEs have most recently become the basis for DSB-independent large targeted insertions ( Yarnall et al, 2022 ), and novel glycosylase-based BEs even permit catalysis of C-to-G transversion events ( Cao et al, 2022 ; Sun et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis

Papaioannou

Patsali²,

Naiisseh³

et al. 2023

Front. Genome Ed.

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Introduction: Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34+ cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for in vitro manipulation. Moreover, ex vivo editing of CD34+ cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies.Methods: Here, we detail an in vitro transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34+ cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal in vitro delivery of genome editing tools.Results and Discussion: Drawing on a common use case, we employ the protocol to target a β-globin mutation and to reactivate γ-globin expression as two potential therapies for β-hemoglobinopathies, followed by erythroid differentiation and functional analyses. Our protocol allows high editing efficiencies and unimpaired cell viability and differentiation, with scalability, suitability for functional assessment of editing outcomes and high flexibility in the application to different editors.

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Section: Introductionmentioning

confidence: 99%

High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis

Papaioannou

Patsali²,

Naiisseh³

et al. 2023

Front. Genome Ed.

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Insights into Prime Editing Technology: A Deep Dive into Fundamentals, Potentials, and Challenges

Hosseini,

Mallick,

Mäkinen

et al. 2024

Human Gene Therapy

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Developing a highly efficient CGBE base editor in watermelon

Wang,

Chen,

Zhu

et al. 2023

Horticulture Research

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Cytosine and adenosine base editors (CBEs and ABEs) are novel genome-editing tools that have been widely utilized in molecular breeding to precisely modify single-nucleotide polymorphisms (SNPs) critical for plant agronomic traits and species evolution. However, conventional BE editors are limited to achieve C-to-T and A-to-G substitutions, respectively. To enhance the applicability of base editing technology in watermelon, we developed an efficient CGBE editor (SCGBE2.0) by removing the UGI unit from the commonly used hA3A-CBE and incorporating the UNG component. Seven specific guide RNAs (sgRNAs) targeting five watermelon genes were designed to assess the editing efficiency of SCGBE. The results obtained from stably transformed watermelon plants demonstrated that SCGBE2.0 could efficiently induce C-to-G mutations at positions C5-C9 in 43.2% transgenic plants (with a maximum base conversion efficiency of 46.1%) and C-to-A mutation at position C4 in 23.5% transgenic plants (with a maximum base conversion efficiency of 45.9%). These findings highlight the capability of our integrated SCGBE2.0 editor to achieve C-to-G/A mutations in a site-preferred manner, thus providing an efficient base editing tool for precise base modification and site-directed saturated mutagenesis in watermelon.

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Generation of C-to-G transversion in mouse embryos via CG editors

Cited by 5 publications

References 45 publications

High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis

High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis

Insights into Prime Editing Technology: A Deep Dive into Fundamentals, Potentials, and Challenges

Developing a highly efficient CGBE base editor in watermelon

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