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Summary Adenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site‐specific A‐to‐G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer‐adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high‐efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single‐stranded DNA‐binding domain (DBD), based on PAM‐less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n‐NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base‐editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e‐DBD‐SpRYn (hyABE8e‐SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)‐dependent off‐target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base‐editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.

Transcription factors (TFs) orchestrate gene expression control of a cell and, in many respects, their repertoire determines the life and functionality of the cell. For a better understanding of their regulatory mechanisms, it is essential to know the entire repertoire of TFs of a species. The increasing number of sequenced genomes together with the development of computational methods allow us not only to predict whole sets of TFs but also to analyse and compare them. Such an analysis is required in particular for fungal species, as our knowledge of the potential set of TFs in fungi is very limited. In fact, at present we do not know which TFs can in general be found in fungi, and which of them are strictly fungal specific. Other interesting questions regard the evolutionary relationships of fungal TFs with other kingdoms and the functions of fungal-specific TFs. This minireview addresses these issues. The analysis of predicted occurrences of DNA-binding domains in 62 fungal genomes reveals a set of 37 potential 'fungal' TF families. Six families are fungal-specific, i.e. they do not appear in other kingdoms. Interestingly, the fungal-specific TFs are not restricted to strictly fungal-specific functions. Consideration of fungal TF distributions in different kingdoms provides a platform to discuss the evolution of domains and TFs.

Background Human papillomavirus (HPV) is a prevalent viral pathogen that causes a variety of malignancies, including cervical cancer, one of the leading causes of cancer-related deaths among women worldwide. The HPV E2 protein is a central regulator of viral replication and oncogene expression, making it a critical determinant of HPV-associated malignancies. While its core functions are conserved, variations within the E2 protein are thought to contribute to the differential oncogenic potential among HPV types, though the structural basis for this remains incompletely understood. Previous research from our laboratory suggests that mutations within a 12–base pair segment of the long control region that encompasses the E2 binding sites may influence the oncogenic potential of certain HPV strains. Methods Computational methods, including multiple sequence alignment, phylogenetic analysis, and protein structural modeling were employed to identify conserved regions and correlate these with potential cancer-associated mutations in the coding region. Results Structural modeling using AlphaFold3 and visualization in PyMOL revealed that conserved E2 residues cluster near the DNA-binding surface in the C-terminal domain and at critical interaction sites in the N-terminal transactivation domain, important for E1 DNA helicase binding and potentially other host factor interactions. Notably, species-specific adaptations, including the T309P substitution in the HPV52 subfamily B2, which may induce structural changes in the DNA-binding domain, and variations in the 12-base pair spacer, could modulate oncogene expression. Conclusions Collectively, these findings refine our understanding of E2’s essential role in viral pathogenesis and highlight promising targets for therapeutic intervention in high-risk HPV strains.

Summary Dual base editors (DBEs) enable simultaneous A‐to‐G and C‐to‐T conversions, expanding mutation types. However, low editing efficiency and narrow targeting range limit the widespread use of DBEs in plants. The single‐strand DNA binding domain of RAD51 DBD can be fused to base editors to improve their editing efficiency. However, it remains unclear how the DBD affects dual base editing performance in plants. In this study, we generated a series of novel plant DBE‐SpGn tools consisting of nine constructs using the high‐activity cytidine deaminase evoFERNY, adenosine deaminase TadA8e and DBD in various fusion modes with the PAM‐flexible Streptococcus pyogenes Cas9 (SpCas9) nickase variant SpGn (with NG‐PAM). By analysing their editing performance on 48 targets in rice, we found that DBE‐SpGn constructs containing a single DBD and deaminases located at the N‐terminus of SpGn exhibited the highest editing efficiencies. Meanwhile, constructs with deaminases located at the C‐terminus and/or multiple DBDs failed to function normally and exhibited inhibited editing activity. We identified three particularly high‐efficiency dual base editors (C‐A‐SpGn, C‐A‐D‐SpGn and A‐C‐D‐SpGn), named PhieDBEs (Plant high‐efficiency dual base editors), capable of producing efficient dual base conversions within a narrow editing window (M5 ~ M9, M = A/C). The editing efficiency of C‐A‐D‐SpGn was as high as 95.2% at certain target sites, with frequencies of simultaneous C‐to‐T and A‐to‐G conversions as high as 81.0%. In summary, PhieDBEs (especially C‐A‐D‐SpGn) can produce diverse mutants and may prove useful in a wide variety of applications, including plant functional genomics, precise mutagenesis, directed evolution and crop genetic improvement, among others.

The transcription factor c-myb has emerged as one of the key regulators of vertebrate hematopoiesis. In mice, it is dispensable for primitive stages of blood cell development but essentially required for definitive hematopoiesis. Using a conditional knockout strategy, recent studies have indicated that c-myb is required for self-renewal of mouse hematopoietic stem cells. Here, we describe and characterize the c-myb mutant in a lower vertebrate, the zebra-fish Danio rerio. The recessive loss-of-function allele of c-myb (c-myb t25127 ) was identified in a collection of N-ethyl-N-nitrosourea (ENU)-induced mutants exhibiting a failure of thymopoiesis. The sequence of the mutant allele predicts a missense mutation (I181N) in the middle of the DNA recognition helix of repeat 3 of the highly conserved DNA binding domain. In keeping with the findings in the mouse, primitive hematopoiesis is not affected in the c-myb mutant fish. By contrast, definitive hematopoiesis fails, resulting in the loss of all blood cells by day 20 of development. Thus, the mutant fish lack lymphocytes and other white and red blood cells; nonetheless, they survive for 2-3 mo but show stunted growth. Because the mutant fish survive into early adulthood, it was possible to directly show that their definitive hematopoiesis is permanently extinguished. Our results, therefore, suggest that the key role of c-myb in definitive hematopoiesis is similar to that in mammals and must have become established early in vertebrate evolution.

The petunia NAM and ArabidopsisATAF1 and CUC2 genes define the conserved NAC domain. In petunia, loss-of-function nam mutants result in embryos that fail to elaborate shoot apical meristems (SAM), and nam seedlings do not develop shoots and leaves. We have isolated a NAC domain gene, AtNAM, from an Arabidopsis developing seed cDNA library. Expression of AtNAM mRNA is restricted primarily to the region of the embryo including the SAM. The AtNAM gene contains three exons and is located on Chromosome 1. In vivo assays in yeast demonstrate that AtNAM encodes a transcription factor and that the NAC domain includes a specific DNA binding domain (DBD). The AtNAM DBD is contained within a 60 amino acid region which potentially folds into a helix-turn-helix motif that specifically binds to the CaMV 35S promoter. The putative transcriptional activation domain is located in the C-terminal region of the protein, a highly divergent region among NAC domain-containing genes. The Arabidopsis genome contains 90 predicted NAC domain genes; we refer to these collectively as the AtNAC superfamily. The first two exons of all members of this superfamily encode the NAC domain. Most AtNAC genes contain three exons with the last exon encoding an activation domain. A subfamily of AtNAC genes contains additional terminal exons coding for protein domains whose functions are unknown.

Generating and applying new knowledge from the wealth of available genomic information is hindered, in part, by the difficulty of altering nucleotide sequences and expression of genes in living cells in a targeted fashion. Progress has been made in engineering DNA binding domains to direct proteins to particular sequences for mutagenesis or manipulation of transcription; however, achieving the requisite specificities has been challenging. Transcription activator—like (TAL) effectors of plant pathogenic bacteria contain a modular DNA binding domain that appears to overcome this challenge. Comprising tandem, polymorphic amino acid repeats that individually specify contiguous nucleotides in DNA, this domain is being deployed in DNA targeting for applications ranging from understanding gene function in model organisms to improving traits in crop plants to treating genetic disorders in people.

Androgens influence transcription of their target genes through the activation of the androgen receptor (AR) that subsequently interacts with specific DNA motifs in these genes. These DNA motifs, called androgen response elements (AREs), can be classified in two classes: the classical AREs, which are also recognized by the other steroid hormone receptors; and the AR-selective AREs, which display selectivity for the AR. For in vitro interaction with the selective AREs, the androgen receptor DNA-binding domain is dependent on specific residues in its second zinc-finger. To evaluate the physiological relevance of these selective elements, we generated a germ-line knockin mouse model, termed SPARKI (SPecificity-affecting AR KnockIn), in which the second zinc-finger of the AR was replaced with that of the glucocorticoid receptor, resulting in a chimeric protein that retains its ability to bind classical AREs but is unable to bind selective AREs. The reproductive organs of SPARKI males are smaller compared with wild-type animals, and they are also subfertile. Intriguingly, however, they do not display any anabolic phenotype. The expression of two testis-specific, androgen-responsive genes is differentially affected by the SPARKI mutation, which is correlated with the involvement of different types of response elements in their androgen responsiveness. In this report, we present the first in vivo evidence of the existence of two functionally different types of AREs and demonstrate that AR-regulated gene expression can be targeted based on this distinction.

In this review, we focus on the assembly of DNA/protein complexes that trigger transposition in eukaryotic members of the IS630-Tc1-mariner (ITm) super-family, the Tc1- and mariner-like elements (TLEs and MLEs). Elements belonging to this super-family encode transposases with DNA binding domains of different origins, and recent data indicate that the chimerization of functional domains has been an important evolutionary aspect in the generation of new transposons within the ITm super-family. These data also reveal that the inverted terminal repeats (ITRs) at the ends of transposons contain three kinds of motif within their sequences. The first two are well known and correspond to the cleavage site on the outer ITR extremities, and the transposase DNA binding site. The organization of ITRs and of the transposase DNA binding domains implies that differing pathways are used by MLEs and TLEs to regulate transposition initiation. These differences imply that the ways ITRs are recognized also differ leading to the formation of differently organized synaptic complexes. The third kind of motif is the transposition enhancers, which have been found in almost all the functional MLEs and TLEs analyzed to date. Finally, in vitro and in vivo assays of various elements all suggest that the transposition initiation complex is not formed randomly, but involves a mechanism of oriented transposon scanning.

The first described feedback loop of the Arabidopsis circadian clock is based on reciprocal regulation between TIMING OF CAB EXPRESSION 1 (TOC1) and CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY). CCA1 and LHY are Myb transcription factors that bind directly to the TOC1 promoter to negatively regulate its expression. Conversely, the activity of TOC1 has remained less well characterized. Genetic data support that TOC1 is necessary for the reactivation of CCA1/LHY, but there is little description of its biochemical function. Here we show that TOC1 occupies specific genomic regions in the CCA1 and LHY promoters. Purified TOC1 binds directly to DNA through its CCT domain, which is similar to known DNA-binding domains. Chemical induction and transient overexpression of TOC1 in Arabidopsis seedlings cause repression of CCA1/LHY expression, demonstrating that TOC1 can repress direct targets, and mutation or deletion of the CCT domain prevents this repression showing that DNA-binding is necessary for TOC1 action. Furthermore, we use the Gal4/UAS system in Arabidopsis to show that TOC1 acts as a general transcriptional repressor, and that repression activity is in the pseudoreceiver domain of the protein. To identify the genes regulated by TOC1 on a genomic scale, we couple TOC1 chemical induction with microarray analysis and identify previously unexplored potential TOC1 targets and output pathways. Taken together, these results define a biochemical action for the core clock protein TOC1 and refine our perspective on how plant clocks function.