A compact dual base editor “A&GBE” is engineer by fusing a deaminase and glycosylase with nickase Cas9 to enable concurrent adenine and cytosine editing and develops quadruple base mutating modules (QBMM) that enables simultaneous mutation of all four types of nucleotide bases.
Abstract
Current dual base editors and hypermutators exhibit limited types of base conversion and constrained mutational diversity. To address this challenge, we engineer a compact dual base editor “A&GBE” by fusing a deaminase and glycosylase with nickase Cas9 (nCas9) to enable concurrent adenine and cytosine editing. Furthermore, we develop quadruple base mutating modules (QBMM) by fusing nickase or dead Cas9 with engineered thymine-DNA glycosylase (TDG), N-methylpurine DNA glycosylase (MPG) and TadA8e, together with MS2-MCP system recruited activation-induced cytidine deaminase (AID). This QBMM enables simultaneous mutation of all four types of nucleotide bases (A, T, G, C) within an approximately 100 base pair (bp) sequence surrounding the gRNA target site. This platform generates hyper diverse multi-nucleotide variants, establishing a powerful tool for accelerated protein evolution, functional genomics, and disease modeling.
Base editors (BEs) enable efficient A-to-G or C-to-T conversions without double-stranded DNA cleavage, but their editing windows remain difficult to tune, limiting genome engineering flexibility. Here, we engineered CRISPR/Cas12b sgRNA by introducing MS2 hairpins to recruit an MS2-N55K-cytidine deaminase-UGI complex, e...
Base editors hold great promise in endogenous mutagenesis for genetic screening. However, the development of base editors that induce saturated multi-base conversions with diverse mutation spectrum is challenging. Here, we develop triple base editors (smACGs) that simultaneously mutagenize adenine, cytosine, and guanin...
This study demonstrates donor-complementary prime editing (DoPE) as a one-step, DSB-free and library-compatible method for precise insertion of large DNA fragments without requiring recombinases or transposases.
Yun-Zheng Fang, Jingyao Tang, Jia-Wei Xi et al.· Nature Biotechnology· 0 citations
MUTATOR is reported, a MUlTiplexAble and self-iTerative ORthogonal base-editing platform that enables N-to-N diversification in Escherichia coli and is established as a broadly applicable platform for genome-wide diversification, functional dissection, and rapid engineering of industrial microbial chassis.
Xiangrui Fan, Li-Ya Liang, Hongle Wang et al.· Nucleic Acids Research· 0 citations
This review compares Cas9-mediated homology-directed repair (HDR) with generations of cytosine base editors (CBE1–CBE3), adenine base editors (ABE1-ABE7), and prime editors (PE1–PE3b), focusing on their mechanistic distinctions, efficiencies, delivery challenges, and therapeutic applications.
Anoushka Sinha· American Journal of Student...· 0 citations
The engineered tBE (etBE) exhibited substantially enhanced editing efficiencies compared with the parental tBE, while maintaining high editing fidelity and background levels of OT mutations, and represents a highly efficient and specific base editing platform with enormous potential for both basic research and clinical...
Bo-Wen Chen, Le-Tong Liang, Ruijuan Xu et al.· Advancement of science· 0 citations
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