PhieABEs: a PAM‐less/free high‐efficiency adenine base editor toolbox with wide target scope in plants

Tan, Jiantao; Zeng, Dongchang; Zhao, Yongchun; Wang, Yaxi; Liu, Taoli; Li, Shuangchun; Yang, Xue; Luo, Yuyu; Xie, Xianrong; Chen, Letian; Liu, Yao‐Guang; Zhu, Qinlong

doi:10.1111/pbi.13774

Cited by 47 publications

(38 citation statements)

References 37 publications

(108 reference statements)

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“…A single-stranded DNA-binding domain from Rad51 protein (RAD51-DBD) was inserted between the deaminases and nCas9, which was reported to substantially increase activity and an expand editing window of CBE ( Zhang et al, 2020 ). Tan et al (2022) recently reported that RAD51-DBD can also dramatically increase editing efficiency of ABE. This CBE system contains a CsU6-tRNA-gRNA-scaffold unit for multiplex editing.…”

Section: Resultsmentioning

confidence: 99%

Base Editors for Citrus Gene Editing

Huang

Wang

2022

Front. Genome Ed.

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Base editors, such as adenine base editors (ABE) and cytosine base editors (CBE), provide alternatives for precise genome editing without generating double-strand breaks (DSBs), thus avoiding the risk of genome instability and unpredictable outcomes caused by DNA repair. Precise gene editing mediated by base editors in citrus has not been reported. Here, we have successfully adapted the ABE to edit the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit (Citrus paradise) and sweet orange (Citrus sinensis). TATA-edited plants are resistant to the canker pathogen Xanthomonas citri subsp. citri (Xcc). In addition, CBE was successfully used to edit the acetolactate synthase (ALS) gene in citrus. ALS-edited plants were resistant to the herbicide chlorsulfuron. Two ALS-edited plants did not show green fluorescence although the starting construct for transformation contains a GFP expression cassette. The Cas9 gene was undetectable in the herbicide-resistant citrus plants. This indicates that the ALS edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression.

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Section: Resultsmentioning

confidence: 99%

Base Editors for Citrus Gene Editing

Huang

Wang

2022

Front. Genome Ed.

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“…Recently, the addition of the human Rad51 DBD to ABE systems produced diverse results in editing efficiency when fused to different Cas9 variants ( Tan et al, 2022 ).…”

Section: Discussionmentioning

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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“…A single-stranded DNA-binding domain from Rad51 protein (RAD51-DBD) was inserted between the deaminases and nCas9, which was reported to substantially increase activity and an expand editing window of CBE (Zhang et al, 2020). Tan et al recently reported that RAD51-DBD can also dramatically increase editing efficiency of ABE (Tan et al, 2022). This CBE system contains a CsU6-tRNA-gRNA-scaffold unit for multiplex editing.…”

Section: Developing a Cbe For Citrusmentioning

confidence: 99%

Base editors for citrus gene editing

Huang

Wang

2021

Preprint

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Base editors, such as adenine base editors (ABE) and cytosine base editors (CBE), provide alternatives for precise genome editing without generating double-strand breaks (DSBs), thus avoiding the risk of genome instability and unpredictable outcomes caused by DNA repair. Precise gene editing mediated by base editors in citrus has not been reported. Here, we have successfully adapted the ABE to modify the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit and Hamlin sweet orange. Inoculation of the TATA-edited plants with the canker pathogen Xanthomonas citri subsp. Citri (Xcc) demonstrated that the TATA-edited plants were resistant to Xcc. In addition, CBE was successfully used to edit the acetolactate synthase (ALS) gene of Carrizo citrange, a hybrid of Citrus sinensis ‘Washington’ sweet orange X Poncirus trifoliata. Editing the ALS genes conferred resistance of Carrizo to the herbicide chlorsulfuron. Two ALS-edited Carrizo plants did not show green florescence although the starting construct for transformation contains a GFP expression cassette. We performed PCR amplification for Cas9 gene in the mutant plants and found that Cas9 gene was undetectable in the herbicide resistant citrus plants. This indicates that the ALS edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression.

show abstract

PhieABEs: a PAM‐less/free high‐efficiency adenine base editor toolbox with wide target scope in plants

Cited by 47 publications

References 37 publications

Base Editors for Citrus Gene Editing

Base Editors for Citrus Gene Editing

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

Base editors for citrus gene editing

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