Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing

Chen, Liang; Zhu, Biyun; Ru, Gaomeng; Meng, Haowei; Yan, Yongchang; Hong, Mengjia; Zhang, Dan; Luan, Changming; Zhang, Shun; Wu, Hao; Gao, Hongyi; Bai, Sijia; Li, Changqing; Ding, Ruoyi; Xue, Niannian; Lei, Zhi‐Xin; Chen, Yuting; Guan, Yuting; Siwko, Stefan; Cheng, Yiyun; Song, Gaojie; Wang, Liren; Yi, Chengqi; Liu, Mingyao; Li, Dali

doi:10.1038/s41587-022-01532-7

Cited by 90 publications

(56 citation statements)

References 46 publications

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“…It is widely believed that complicated engineering of adenosine deaminases via directed protein evolution was necessary to generate functional ABEs and all available ABEs were derived from ecTadA with more than ten amino acid substitutions 2 . Here we demonstrated that in combined with an internal fusion strategy at specific insertion sites of nCas9, engineering of ecTadA with just two amino acid substitutions at conserved residues could generate functional ABEs with obvious adenine editing activity and detectable cytosine editing activity, consistent with previous studies showing that classical ABEs containing evolved ecTadA displayed cytosine editing activity within specific sequence contexts 18,[28][29][30][31] . By screening tens of different TadA orthologs in HEK293T cells with flow cytometry (Supplementary Fig.…”

Section: Discussionsupporting

confidence: 90%

TadA orthologs enable both cytosine and adenine editing of base editors

et al. 2023

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Cytidine and adenosine deaminases are required for cytosine and adenine editing of base editors respectively, and no single deaminase could enable concurrent and comparable cytosine and adenine editing. Additionally, distinct properties of cytidine and adenosine deaminases lead to various types of off-target effects, including Cas9-indendepent DNA off-target effects for cytosine base editors (CBEs) and RNA off-target effects particularly severe for adenine base editors (ABEs). Here we demonstrate that 25 TadA orthologs could be engineered to generate functional ABEs, CBEs or ACBEs via single or double mutations, which display minimized Cas9-independent DNA off-target effects and genotoxicity, with orthologs B5ZCW4, Q57LE3, E8WVH3, Q13XZ4 and B3PCY2 as promising candidates for further engineering. Furthermore, RNA off-target effects of TadA ortholog-derived base editors could be further reduced or even eliminated by additional single mutation. Taken together, our work expands the base editing toolkits, and also provides important clues for the potential evolutionary process of deaminases.

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Section: Discussionsupporting

confidence: 90%

TadA orthologs enable both cytosine and adenine editing of base editors

et al. 2023

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“…In the future, the use of engineered deaminase domains with reduced Cas9-independent off-targeting 50,51 might further reduce the toxicity of base editors in Drosophila and other species. Likewise, recently described TadA-based CBEs have been shown to minimise the occurrence of DNA and RNA off-targeting 52,53 .…”

Section: Discussionmentioning

confidence: 97%

A temperature-tolerant CRISPR base editor mediates highly efficient and precise gene inactivationin vivo

Doll

Boutros

Port

2022

Preprint

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CRISPR nucleases generate a broad spectrum of mutations that includes undesired editing outcomes which attenuate phenotypes and complicate experimental analysis and interpretation. Here, we develop an optimised cytosine base editing system for gene inactivation in Drosophila through predictable C-to-T editing and identify temperature as a crucial parameter for base editing efficiency. We find that activity of an evolved version of the most widely used APOBEC1 deaminase is attenuated within the temperature range commonly used for culturing Drosophila (18-29 degrees C) and many other ectothermic species. In contrast, an evolved CDA1 domain functions with remarkable efficiency within the same temperature range. Furthermore, we show that formation of undesired indel mutations and C-to-G/A edits is exceptionally rare in Drosophila compared to other species. The predictable editing outcome, very high efficiency and minimal byproduct formation of this system allows for near homogeneous biallelic gene inactivation in vivo in a ubiquitous or conditional manner. This work significantly improves our ability to create precise loss-of-function alleles in Drosophila and provides key design parameters for developing highly efficient base editing systems in other ectothermic species.

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“…Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination, representing the first unnatural cytosine deaminases besides the natural AID/APOBEC protein family. Briefly, Td-CGBE performs highly efficient and precise C-to-G editing 175 , while Td-CBEs 175 and TadCBEs 174 generate precise Cto-T base conversions with the fusion of UGI and additional mutations. Moreover, TadA dual-base editor (TadDE) was shown to perform equally efficient cytosine and adenine base editing concurrently (C-to-T and A-to-G) 174 , providing a highly efficient and small size alternative besides the previously reported dual editors that fuse both cytidine and adenosine deaminases (ACBEs) [137][138][139][140][141] , as well as enabling saturation mutagenesis and screening.…”

Section: Methods For Reducing Off-target Effects Of Dna Base Editingmentioning

confidence: 99%

“…Since ABEs show lower DNA and RNA off-target activity, smaller size and higher on-target editing efficiency than CBEs, two groups re-engineered ABEs for efficient and specific CRISPR-based cytosine base editing 174 , 175 . Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination, representing the first unnatural cytosine deaminases besides the natural AID/APOBEC protein family.…”

Section: Dna Base Editorsmentioning

confidence: 99%

Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing

2023

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CRISPR-Cas gene editing has revolutionized experimental molecular biology over the past decade and holds great promise for the treatment of human genetic diseases. Here we review the development of CRISPR-Cas9/Cas12/Cas13 nucleases, DNA base editors, prime editors, and RNA base editors, focusing on the assessment and improvement of their editing precision and safety, pushing the limit of editing specificity and efficiency. We summarize the capabilities and limitations of each CRISPR tool from DNA editing to RNA editing, and highlight the opportunities for future improvements and applications in basic research, as well as the therapeutic and clinical considerations for their use in patients.

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Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing

Cited by 90 publications

References 46 publications

TadA orthologs enable both cytosine and adenine editing of base editors

TadA orthologs enable both cytosine and adenine editing of base editors

A temperature-tolerant CRISPR base editor mediates highly efficient and precise gene inactivationin vivo

Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing

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