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

Song, Min Kyoung; Lim, Jung Min; Min, Shungeng; Oh, Jae-Ho; Kim, Dong Young; Woo, Jae Sung; Nishimasu, Hiroshi; Cho, Sung Rae; Yoon, Sungroh; Kim, Hyongbum

doi:10.1038/s41467-021-25928-2

Cited by 60 publications

(35 citation statements)

References 37 publications

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“…1 a). Unlike the higher efficiency of DBD-integrated hyPE2 relative to PE2 in human cells [ 12 ], we found that phyPE2 conducted less efficient editing than pPE2 at the tested sites in rice (Fig. 1 c).…”

Section: Resultscontrasting

confidence: 66%

Development of a highly efficient prime editor 2 system in plants

Chen

Liang

et al. 2022

Genome Biol

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Low efficiency has seriously restricted the application of prime editing (PE) systems in plants. In this study, we develop an enhanced plant prime editor 2 system, enpPE2, by stacking various optimization strategies, including updating the PE architecture to PEmax and expressing engineered pegRNA with a structured motif under the control of a composite promoter. In T0 rice plants, enpPE2 exhibits editing frequencies of 64.58% to 77.08%, which are much higher than the frequencies with unmodified pPE2. Our results indicate that the enpPE2 system provides a robust and powerful tool for the precise modification of plant genomes.

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Section: Resultscontrasting

confidence: 66%

Development of a highly efficient prime editor 2 system in plants

Chen

Liang

et al. 2022

Genome Biol

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“… 31 Moreover, several important progresses have also been made in enhancing the activity of prime editor itself. Equipping the prime editor with strong nuclear localization signal, 32 modifying the 3′ engineered pegRNA to reduce self-complementing 33 or degradation, 34 and enhancing the RT with ssDNA binding domain 35 were all capable of improving prime editing. It is, therefore, expectable that the introduction of these findings to WT-PE system may also increase its editing efficiency.…”

Section: Discussionmentioning

confidence: 99%

WT-PE: Prime editing with nuclease wild-type Cas9 enables versatile large-scale genome editing

Tao

Wang

et al. 2022

Sig Transduct Target Ther

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Large scale genomic aberrations including duplication, deletion, translocation, and other structural changes are the cause of a subtype of hereditary genetic disorders and contribute to onset or progress of cancer. The current prime editor, PE2, consisting of Cas9-nickase and reverse transcriptase enables efficient editing of genomic deletion and insertion, however, at small scale. Here, we designed a novel prime editor by fusing reverse transcriptase (RT) to nuclease wild-type Cas9 (WT-PE) to edit large genomic fragment. WT-PE system simultaneously introduced a double strand break (DSB) and a single 3′ extended flap in the target site. Coupled with paired prime editing guide RNAs (pegRNAs) that have complementary sequences in their 3′ terminus while target different genomic regions, WT-PE produced bi-directional prime editing, which enabled efficient and versatile large-scale genome editing, including large fragment deletion up to 16.8 megabase (Mb) pairs and chromosomal translocation. Therefore, our WT-PE system has great potential to model or treat diseases related to large-fragment aberrations.

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“…In addition, a fusion of the Rad51 DBD allowed editing of target sites that could not be edited using conventional base editors in rice ( Xu et al, 2022 ). Since the Rad51 DBD is a non-sequence-specific protein, it has also been used to stabilize the ssDNA and improve the efficiency in the Primer Editor (PE) system ( Song et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

“…Fusion of the human Rad51 with the Cas9 (D10A) nickase successfully mediated recombination by the homology-directed repair (HDR) pathway ( Rees et al, 2019 ). The Rad51 N-terminal domain contains 114 residues involved in DNA binding ( Aihara et al, 1999 ) and several groups have successfully used this domain with CBEs and prime editors (PEs) to improve editing efficiency and expand the editing window ( Zhang et al, 2020 ; Song et al, 2021 ). Fusion of the Rad51 DBD with the Nmcas9-based CBE, termed hyper CBE (hyCBE), has proven successful in rice.…”

Section: Introductionmentioning

confidence: 99%

Expanding the Editing Window of Cytidine Base Editors With the Rad51 DNA-Binding Domain in Rice

et al. 2022

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Recently developed base editors provide a powerful tool for plant research and crop improvement. Although a number of different deaminases and Cas proteins have been used to improve base editors the editing efficiency, and editing window are still not optimal. Fusion of a non-sequence-specific single-stranded DNA-binding domain (DBD) from the human Rad51 protein between Cas9 nickase and the deaminase has been reported to dramatically increase the editing efficiency and expand the editing window of base editors in the mammalian cell lines and mouse embryos. We report the use of this strategy in rice, by fusing a rice codon-optimized human Rad51 DBD to the cytidine base editors AncBE4max, AncBE4max-NG, and evoFERNY. Our results show that the addition of Rad51 DBD did not increase editing efficiency in the major editing window but the editing range was expanded in all the three systems. Replacing the human Rad51 DBD with the rice Rad51 DBD homolog also expanded the editing window effectively.

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

Cited by 60 publications

References 37 publications

Development of a highly efficient prime editor 2 system in plants

Development of a highly efficient prime editor 2 system in plants

WT-PE: Prime editing with nuclease wild-type Cas9 enables versatile large-scale genome editing

Expanding the Editing Window of Cytidine Base Editors With the Rad51 DNA-Binding Domain in Rice

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