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DNA base editors enable direct editing of adenine (A), cytosine (C), or guanine (G), but there is no base editor for direct thymine (T) editing currently. Here we develop two deaminase-free glycosylase-based base editors for direct T editing (gTBE) and C editing (gCBE) by fusing Cas9 nickase (nCas9) with engineered human uracil DNA glycosylase (UNG) variants. By several rounds of structure-informed rational mutagenesis on UNG in cultured human cells, we obtain gTBE and gCBE with high activity of T-to-S (i.e., T-to-C or T-to-G) and C-to-G conversions, respectively. Furthermore, we conduct parallel comparison of gTBE/gCBE with those recently developed using other protein engineering strategies, and find gTBE/gCBE show the outperformance. Thus, we provide several base editors, gTBEs and gCBEs, with corresponding engineered UNG variants, broadening the targeting scope of base editors. Efficient base editors for direct thymine (T) editing are highly desirable. Here, authors develop two deaminase-free glycosylase-based base editors for direct T editing (gTBE) and C editing (gCBE) by rounds of structure-informed mutagenesis on human DNA glycosylase UNG and further engineering.

Uracil DNA glycosylases (UDGs) are important DNA repair enzymes that excise uracil from DNA, yielding an abasic site. Recently, UdgX, an unconventional UDG with extremely tight binding to DNA containing uracil, was discovered. The structure of UdgX from Mycobacterium smegmatis in complex with DNA shows an overall similarity to that of family 4 UDGs except for a protruding loop at the entrance of the uracil-binding pocket. Surprisingly, H109 in the loop was found to make a covalent bond to the abasic site to form a stable intermediate, while the excised uracil remained in the pocket of the active site. H109 functions as a nucleophile to attack the oxocarbenium ion, substituting for the catalytic water molecule found in other UDGs. To our knowledge, this change from a catalytic water attack to a direct nucleophilic attack by the histidine residue is unprecedented. UdgX utilizes a unique mechanism of protecting cytotoxic abasic sites from exposure to the cellular environment. Structural analysis of uracil DNA glycosylases in complex with DNA reveals that conserved H109 acts as a nucleophile to attack the oxocarbenium ion and makes a covalent bond to the abasic site after uracil excision to form a stable intermediate.

Dear Editor, Base editors （BEs） have been recently developed by combining the APOBEC （apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like）/AID （acti- vation-induced deaminase） cytidine deaminase family members [1] with the CRISPR./Cas9 system to perform targeted C-to-T base editing [2-8]. Mechanistically, Cas9 variant-fused APOBEC/AID is directed to target site by sgRNA, introducing C-to-T substitution at the single-base level [2-4]. Compared to earlier generations of BEs （BE1 and BE2）, the latest BE3 achieved much higher base editing frequencies by substituting catalyti- cally-dead Cas9 （dCas9） with Cas9 nickase （nCas9） [2].

Ricin can be isolated from the seeds of the castor bean plant (Ricinus communis). It belongs to the ribosome-inactivating protein (RIP) family of toxins classified as a bio-threat agent due to its high toxicity, stability and availability. Ricin is a typical A-B toxin consisting of a single enzymatic A subunit (RTA) and a binding B subunit (RTB) joined by a single disulfide bond. RTA possesses an RNA N-glycosidase activity; it cleaves ribosomal RNA leading to the inhibition of protein synthesis. However, the mechanism of ricin-mediated cell death is quite complex, as a growing number of studies demonstrate that the inhibition of protein synthesis is not always correlated with long term ricin toxicity. To exert its cytotoxic effect, ricin A-chain has to be transported to the cytosol of the host cell. This translocation is preceded by endocytic uptake of the toxin and retrograde traffic through the trans-Golgi network (TGN) and the endoplasmic reticulum (ER). In this article, we describe intracellular trafficking of ricin with particular emphasis on host cell factors that facilitate this transport and contribute to ricin cytotoxicity in mammalian and yeast cells. The current understanding of the mechanisms of ricin-mediated cell death is discussed as well. We also comment on recent reports presenting medical applications for ricin and progress associated with the development of vaccines against this toxin.

Plants contain numerous glycoconjugates that are metabolized by specific glucosyltransferases and hydrolyzed by specific glycosidases, some also catalyzing synthetic transglycosylation reactions. The documented value of plant-derived glycoconjugates to beneficially modulate metabolism is first addressed. Next, focus is given to glycosidases, the central theme of the review. The therapeutic value of plant glycosidases is discussed as well as the present production in plant platforms of therapeutic human glycosidases used in enzyme replacement therapies. The increasing knowledge on glycosidases, including structure and catalytic mechanism, is described. The novel insights have allowed the design of functionalized highly specific suicide inhibitors of glycosidases. These so-called activity-based probes allow unprecedented visualization of glycosidases cross-species. Here, special attention is paid on the use of such probes in plant science that promote the discovery of novel enzymes and the identification of potential therapeutic inhibitors and chaperones.

Enzymes in uracil-DNA glycosylase (UDG) superfamily are involved in removal of deaminated nucleobases such as uracil, methylcytosine derivatives such as formylcytosine and carboxylcytosine, and other base damage in DNA repair. UDGX is the latest addition of a new class to the UDG superfamily with a sporadic distribution in bacteria. UDGX type enzymes have a distinct biochemical property of cross-linking itself to the resulting AP site after uracil removal. Built on previous biochemical and structural analyses, this work comprehensively investigated the kinetic and enzymatic properties of Mycobacterium smegmatis UDGX. Kinetics and mutational analyses, coupled with structural information, defined the roles of E52, D56, D59, F65 of motif 1, H178 of motif 2 and N91, K94, R107 and H109 of motif 3 play in uracil excision and cross-linking. More importantly, a series of quantitative analyses underscored the structural coupling through inter-motif and intra-motif interactions and subsequent functional coupling of the uracil excision and cross-linking reactions. A catalytic model is proposed, which underlies this catalytic feature unique to UDGX type enzymes. This study offers new insight on the catalytic mechanism of UDGX and provides a unique example of enzyme evolution.

A uracil DNA glycosylase (UDG) from Mycobacterium smegmatis ( Msm UdgX) shares sequence similarity with family 4 UDGs and forms exceedingly stable complexes with single-stranded uracil-containing DNAs (ssDNA-Us) that are resistant to denaturants. However, Msm UdgX has been reported to be inactive in excising uracil from ssDNA-Us and the underlying structural basis is unclear. Here, we report high-resolution crystal structures of Msm UdgX in the free, uracil- and DNA-bound forms, respectively. The structural information, supported by mutational and biochemical analyses, indicates that the conserved residue His109 located on a characteristic loop forms an irreversible covalent linkage with the deoxyribose at the apyrimidinic site of ssDNA-U, thus rendering the enzyme unable to regenerate. By proposing the catalytic pathway and molecular mechanism for Msm UdgX, our studies provide an insight into family 4 UDGs and UDGs in general. Structural analysis of uracil DNA glycosylase reveals that its high affinity with DNA substrates derives from a stable intermediate that is formed by conservative H109 in a protruding loop covalently binding to the abasic site after uracil is excised.

A cod uracil DNA glycosylase from the cold-adapted organism Gadus morhua enhanced cytosine (C)-to-guanine (G) base editor (CGBE) activities in rice.TadA-8e-derived cytidine deaminase (TadA-CDc) variants enabled rice C-to-G conversions.CDc-CGBEco achieved highly efficient C-to-G editing in rice, soybean, and tobacco.No significant off-target effects of the base editors derived from TadA-CDc were detected in rice. Plant cytosine (C)-to-guanine (G) base editors (CGBEs) have been established but suffer from limited editing efficiencies and low outcome purities. This study engineered a cod uracil DNA glycosylase (cod UNG, coUNG) from the cold-adapted fish Gadus morhua for plant CGBE, demonstrating 1.71- to 2.54-fold increases in C-to-G editing efficiency compared with the CGBE using human UNG (hUNG). Further engineering took advantage of TadA-8e-derived cytidine deaminases (TadA-CDs). These variants induced C substitutions with efficiencies ranging from 26.28% to 30.82% in rice cells, whereas adenine (A) conversion was negligible. By integrating coUNG and TadA-CDc elements with SpCas9 nickase, the resulting CDc-CGBEco achieved pure C-to-G editing without byproducts in up to 52.08% of transgenic lines. Whole-genome sequencing (WGS) analysis revealed no significant off-target effects of the CDc-BEs in rice. In addition, CDc-CGBEco enabled precise C-to-G editing in soybean and tobacco. These engineered CGBEs enhanced editing efficiency, purity, and specificity, suggesting their broad potential for applications in scientific research and crop breeding. [Display omitted] CDc-CGBEco introduced in this study is currently at a Technology Readiness Level (TRL) of 5. Using CDc-CGBEco, we obtained C-to-G-edited T0 rice plants with improved agronomic traits, such as herbicide resistance; we also found germline transmission of pure C-to-G edits in T1 offspring in the field. However, several challenges remain. For instance, limited editing activity in tobacco suggests that the universal applicability of CDc-CGBEco needs to be enhanced. In addition, although whole-genome sequencing was conducted to assess off-target effects, more long-term monitoring and diverse condition evaluations are necessary to comprehensively verify the safety and stability of this technology. A series of uracil DNA glycosylases (UNG) and cytidine deaminases were screened for upgrading plant cytosine (C)-to-guanine (G) base editors (CGBE). An engineered CDc-CGBEco fusing with a cod UNG (coUNG) and a TadA-8e-derived cytidine deaminases (TadA-CDc) exhibited superior performance in C-to-G editing, showing reliable and heritable base editing in monocots and dicots.

Compound K (C-K) is a crucial pharmaceutical and cosmetic component because of disease prevention and skin anti-aging effects. For industrial application of this active compound, the protopanaxadiol (PPD)-type ginsenosides should be transformed to C-K. [beta]-Glycosidase from Sulfolobus solfataricus has been reported as an efficient C-K-producing enzyme, using glycosylated PPD-type ginsenosides as substrates. [beta]-Glycosidase from S. solfataricus can hydrolyze [beta]-d-glucopyranoside in ginsenosides Rc, C-Mc.sub.1, and C-Mc, but not [alpha]-l-arabinofuranoside in these ginsenosides. To determine candidate residues involved in [alpha]-l-arabinofuranosidase activity, compound Mc (C-Mc) was docking to [beta]-glycosidase from S. solfataricus in homology model and sequence was aligned with [beta]-glycosidase from Pyrococcus furiosus that has [alpha]-l-arabinofuranosidase activity. A L213A variant [beta]-glycosidase with increased [alpha]-l-arabinofuranosidase activity was selected by substitution of other amino acids for candidate residues. The increased [alpha]-l-arabinofuranosidase activity of the L213A variant was confirmed through the determination of substrate specificity, change in binding energy, transformation pathway, and C-K production from ginsenosides Rc and C-Mc. The L213A variant [beta]-glycosidase catalyzed the conversion of Rc to Rd by hydrolyzing [alpha]-l-arabinofuranoside linked to Rc, whereas the wild-type [beta]-glycosidase did not. The variant enzyme converted ginsenosides Rc and C-Mc into C-K with molar conversions of 97%, which were 1.5- and 2-fold higher, respectively, than those of the wild-type enzyme. Therefore, protein engineering is a useful tool for enhancing the hydrolytic activity on specific glycoside linked to ginsenosides.

DNA base editing technologies predominantly utilize engineered deaminases, limiting their ability to edit thymine and guanine directly. In this study, we successfully achieve base editing of both cytidine and thymine by leveraging the translesion DNA synthesis pathway through the engineering of uracil-DNA glycosylase (UNG). Employing structure-based rational design, exploration of homologous proteins, and mutation screening, we identify a Deinococcus radiodurans UNG mutant capable of effectively editing thymine. When fused with the nickase Cas9, the engineered DrUNG protein facilitates efficient thymine base editing at endogenous sites, achieving editing efficiencies up to 55% without enrichment and exhibiting minimal cellular toxicity. This thymine base editor (TBE) exhibits high editing specificity and significantly restores IDUA enzyme activity in cells derived from patients with Hurler syndrome. TBEs represent efficient, specific, and low-toxicity approaches to base editing with potential applications in treating relevant diseases. Base editing technologies predominantly utilize engineered deaminases for cytosine and adenine. Here, the authors achieve efficient, specific, and low-toxicity base editing of thymine by engineering Deinococcus radiodurans uracil-DNA glycosylase.