Improving adenine and dual base editors through introduction of TadA-8e and Rad51DBD

Xue, Niannian; Liu, Xu; Zhang, Dan; Wu, Youming; Zhong, Yi; Fan, Wenjing; Jiang, Haixia; Zhu, Biyun; Ge, Xiyu; Gonzalez, Rachel V L; Chen, Liang; Zhang, Shun; She, Peilu; Zhong, Zhimei; Sun, Jie; Chen, Xi; Wang, Liren; Gu, Zhimin; Zhu, Ping; Liu, Mingyao; Li, Dali; Zhong, Tao P; Zhang, Xiaohui

doi:10.1038/s41467-023-36887-1

Cited by 12 publications

(6 citation statements)

References 31 publications

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“…Finally, the rate of on-target indels was estimated to be ≤1.2% for all target loci (0.6, 1.2 and 0.2% for FLT3 , CD123 and KIT , respectively) (Extended Data Fig. 8d ), consistent with previously reported data for ABE 42 , 43 . Overall, these data support a generally safe genotoxicity profile of FLT3, CD123 and KIT epitope editing in CD34 + HSPCs.…”

Section: Off-target Effects Of Epitope Editingsupporting

confidence: 90%

Epitope editing enables targeted immunotherapy of acute myeloid leukaemia

Casirati,

Cosentino,

Mucci

et al. 2023

Nature

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Despite the considerable efficacy observed when targeting a dispensable lineage antigen, such as CD19 in B cell acute lymphoblastic leukaemia1,2, the broader applicability of adoptive immunotherapies is hampered by the absence of tumour-restricted antigens3–5. Acute myeloid leukaemia immunotherapies target genes expressed by haematopoietic stem/progenitor cells (HSPCs) or differentiated myeloid cells, resulting in intolerable on-target/off-tumour toxicity. Here we show that epitope engineering of donor HSPCs used for bone marrow transplantation endows haematopoietic lineages with selective resistance to chimeric antigen receptor (CAR) T cells or monoclonal antibodies, without affecting protein function or regulation. This strategy enables the targeting of genes that are essential for leukaemia survival regardless of shared expression on HSPCs, reducing the risk of tumour immune escape. By performing epitope mapping and library screenings, we identified amino acid changes that abrogate the binding of therapeutic monoclonal antibodies targeting FLT3, CD123 and KIT, and optimized a base-editing approach to introduce them into CD34+ HSPCs, which retain long-term engraftment and multilineage differentiation ability. After CAR T cell treatment, we confirmed resistance of epitope-edited haematopoiesis and concomitant eradication of patient-derived acute myeloid leukaemia xenografts. Furthermore, we show that multiplex epitope engineering of HSPCs is feasible and enables more effective immunotherapies against multiple targets without incurring overlapping off-tumour toxicities. We envision that this approach will provide opportunities to treat relapsed/refractory acute myeloid leukaemia and enable safer non-genotoxic conditioning.

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Section: Off-target Effects Of Epitope Editingsupporting

confidence: 90%

Epitope editing enables targeted immunotherapy of acute myeloid leukaemia

Casirati,

Cosentino,

Mucci

et al. 2023

Nature

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“…It has been reported that fusing a single-strand DNA-binding domain from RADIATION SENSITIVE 51 (Rad51) to a cytosine base editor [ 26 ] or adenine base editor [ 27 , 28 ] could enhance the base editing capability in mammalian cells and rice. We wondered if the fusion of Rad51 could influence the editing efficiency of TALE-DdCBEs; therefore, we inserted the Rad51 domain before the DddA halves or before the TALE N-terminal domain (Additional file 1 : Fig.…”

Section: Resultsmentioning

confidence: 99%

Targeted C•G-to-T•A base editing with TALE-cytosine deaminases in plants

Zhang,

Pries,

Boch

2024

BMC Biol

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Background TALE-derived DddA-based cytosine base editors (TALE-DdCBEs) can perform efficient base editing of mitochondria and chloroplast genomes. They use transcription activator-like effector (TALE) arrays as programmable DNA-binding domains and a split version of the double-strand DNA cytidine deaminase (DddA) to catalyze C•G-to-T•A editing. This technology has not been optimized for use in plant cells. Results To systematically investigate TALE-DdCBE architectures and editing rules, we established a β-glucuronidase reporter for transient assays in Nicotiana benthamiana. We show that TALE-DdCBEs function with distinct spacer lengths between the DNA-binding sites of their two TALE parts. Compared to canonical DddA, TALE-DdCBEs containing evolved DddA variants (DddA6 or DddA11) showed a significant improvement in editing efficiency in Nicotiana benthamiana and rice. Moreover, TALE-DdCBEs containing DddA11 have broader sequence compatibility for non-TC target editing. We have successfully regenerated rice with C•G-to-T•A conversions in their chloroplast genome, as well as N. benthamiana with C•G-to-T•A editing in the nuclear genome using TALE-DdCBE. We also found that the spontaneous assembly of split DddA halves can cause undesired editing by TALE-DdCBEs in plants. Conclusions Altogether, our results refined the targeting scope of TALE-DdCBEs and successfully applied them to target the chloroplast and nuclear genomes. Our study expands the base editing toolbox in plants and further defines parameters to optimize TALE-DdCBEs for high-fidelity crop improvement.

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“…In the second-generation DuBEs, ABE7.10 was replaced with high-performing monomeric ABE variants to increase the likelihood of greater simultaneous A-to-G and C-to-T edits. Notably, TadA-8e (ABE8e) adoption in DuBEs showed higher concurrent BE activities in distinctive DuBE architectures developed by various research teams, such as iACBEs ( 42 ), ACBE ( 43 ), CABE-RY ( 44 ), ACEs ( 5 ), Dual BEs ( 45 ), and hyA&C-BEmax ( 46 ) ( Table 1 ). Recently, ACBE variants without the UGI domain or with the UGI domain replaced by uracil-DNA glycosylase (UNG) have shown increased randomization of deaminated Cs into all possible outcomes (C-to-G/T/A) along with A-to-G and indels, termed as AGBEs ( 47 ).…”

Section: Be-trm Toolbox For Directed Evolutionmentioning

confidence: 99%

CRISPR base editor-based targeted random mutagenesis (BE-TRM) toolbox for directed evolution

Shelake,

Pramanik,

Kim

2024

BMB Rep

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Directed evolution (DE) of desired locus by targeted random mutagenesis (TRM) tools is a powerful approach for generating genetic variations with novel or improved functions, particularly in complex genomes. TRM-based DE involves developing a mutant library of targeted DNA sequences and screening the variants for the desired properties. However, DE methods have for a long time been confined to bacteria and yeasts. Lately, CRISPR/Cas and DNA deaminase-based tools that circumvent enduring barriers such as longer life cycle, small library sizes, and low mutation rates have been developed to facilitate DE in native genetic environments of multicellular organisms. Notably, deaminase-based base editing-TRM (BE-TRM) tools have greatly expanded the scope and efficiency of DE schemes by enabling base substitutions and randomization of targeted DNA sequences. BE-TRM tools provide a robust platform for the continuous molecular evolution of desired proteins, metabolic pathway engineering, creation of a mutant library of desired locus to evolve novel functions, and other applications, such as predicting mutants conferring antibiotic resistance. This review provides timely updates on the recent advances in BE-TRM tools for DE, their applications in biology, and future directions for further improvements.

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Improving adenine and dual base editors through introduction of TadA-8e and Rad51DBD

Cited by 12 publications

References 31 publications

Epitope editing enables targeted immunotherapy of acute myeloid leukaemia

Epitope editing enables targeted immunotherapy of acute myeloid leukaemia

Targeted C•G-to-T•A base editing with TALE-cytosine deaminases in plants

CRISPR base editor-based targeted random mutagenesis (BE-TRM) toolbox for directed evolution

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