Hypercompact adenine base editors based on transposase B guided by engineered RNA.

Transposon-associated transposase B (TnpB) is deemed an ancestral protein for type V, Cas12 family members, and the closest ancestor to UnCas12f1. Previously, we reported a set of engineered guide RNAs supporting high indel efficiency for Cas12f1 in human cells. Here we suggest a new technology whereby the engineered guide RNAs also manifest high-efficiency programmable endonuclease activity for TnpB. We have termed this technology TaRGET (TnpB-augment RNA-based Genome Editing Technology). Having this feature in mind, we established TnpB-based adenine base editors (ABEs). A Tad-Tad mutant (V106W, D108Q) dimer fused to the C terminus of dTnpB (D354A) showed the highest levels of A-to-G conversion. The limited targetable sites for TaRGET-ABE were expanded with engineered variants of TnpB or optimized deaminases. Delivery of TaRGET-ABE also ensured potent A-to-G conversion rates in mammalian genomes. Collectively, the TaRGET-ABE will contribute to improving precise genome-editing tools that can be delivered by adeno-associated viruses, thereby harnessing the development of clustered regularly interspaced short palindromic repeats (CRISPR)-based gene therapy.

Auxin Response Factors promote organogenesis by chromatin-mediated repression of the pluripotency gene SHOOTMERISTEMLESS.

Specification of new organs from transit amplifying cells is critical for higher eukaryote development. In plants, a central stem cell pool maintained by the pluripotency factor SHOOTMERISTEMLESS (STM), is surrounded by transit amplifying cells competent to respond to auxin hormone maxima by giving rise to new organs. Auxin triggers flower initiation through Auxin Response Factor (ARF) MONOPTEROS (MP) and recruitment of chromatin remodelers to activate genes promoting floral fate. The contribution of gene repression to reproductive primordium initiation is poorly understood. Here we show that downregulation of the STM pluripotency gene promotes initiation of flowers and uncover the mechanism for STM silencing. The ARFs ETTIN (ETT) and ARF4 promote organogenesis at the reproductive shoot apex in parallel with MP via histone-deacetylation mediated transcriptional silencing of STM. ETT and ARF4 directly repress STM, while MP acts indirectly, through its target FILAMENTOUS FLOWER (FIL). Our data suggest that - as in animals- downregulation of the pluripotency program is important for organogenesis in plants.

Antagonistic regulation of Arabidopsis growth by brassinosteroids and abiotic stresses.

To withstand ever-changing environmental stresses, plants are equipped with phytohormone-mediated stress resistance mechanisms. Salt stress triggers abscisic acid (ABA) signaling, which enhances stress tolerance at the expense of growth. ABA is thought to inhibit the action of growth-promoting hormones, including brassinosteroids (BRs). However, the regulatory mechanisms that coordinate ABA and BR activity remain to be discovered. We noticed that ABA-treated seedlings exhibited small, round leaves and short roots, a phenotype that is characteristic of the BR signaling mutant, brassinosteroid insensitive1-9 (bri1-9). To identify genes that are antagonistically regulated by ABA and BRs, we examined published Arabidopsis microarray data sets. Of the list of genes identified, those upregulated by ABA but downregulated by BRs were enriched with a BRRE motif in their promoter sequences. After validating the microarray data using quantitative RT-PCR, we focused on RD26, which is induced by salt stress. Histochemical analysis of transgenic Arabidopsis plants expressing RD26pro:GUS revealed that the induction of GUS expression after NaCl treatment was suppressed by co-treatment with BRs, but enhanced by co-treatment with propiconazole, a BR biosynthetic inhibitor. Similarly, treatment with bikinin, an inhibitor of BIN2 kinase, not only inhibited RD26 expression, but also reduced the survival rate of the plant following exposure to salt stress. Our results suggest that ABA and BRs act antagonistically on their target genes at or after the BIN2 step in BR signaling pathways, and suggest a mechanism by which plants fine-tune their growth, particularly when stress responses and growth compete for resources.

Constitutive activation of brassinosteroid signaling in the Arabidopsis elongated-D/bak1 mutant.

Defects in brassinosteroid (BR) biosynthetic or signaling genes result in dwarfed plants, whereas overexpression of these genes increases overall stature. An Arabidopsis elongated-D (elg-D) mutant shares phenotypic similarities with BR overexpression lines, suggesting its implication in BR pathways. Here, we determine how elg-D affects BR signaling. Since elg-D rescued dwarfism in bri1-5 plants, a BR receptor mutant, but not in BR-insensitive bin2/dwf12-1D plants, elg-D appears to act between bri1-5 and bin2/dwf12-1D in BR signaling. We found that elg-D had an increased response to epi-brassinolide (epi-BL); that the BES1 transcription factor was shifted toward the dephosphorylated form in elg-D; that the expression of a BR responsive gene, SAUR-AC1, was upregulated in elg-D; and that transcription of BR biosynthetic genes, DWF4 and CPD, was downregulated by feedback inhibition. Thus, endogenous levels of CS and BL as well as biosynthetic intermediates were reduced by the elg-D mutation, whereas basal levels of BR signaling were elevated. Map-based cloning and sequencing revealed that elg-D is allelic to the BR co-receptor protein, BAK1, and has an Asp(122) to Asn substitution in the third repeat of the extracellular leucine-rich repeat (LRR) domain. In agreement with the finding that BAK1/ELG is involved in the perception of pathogen-associated molecular patterns (PAMPs), the bak1/elg-D plants exhibited increased Pseudomonas syringae growth. Therefore, bak1/elg-D promotes Arabidopsis growth by stimulating BR signaling at the expense of its readiness to respond to biotic stress factors. The BAK1/ELG BR co-receptor thus plays an important role in BR signaling that is mediated by its LRR domain.

Auxin stimulates DWARF4 expression and brassinosteroid biosynthesis in Arabidopsis.

Brassinosteroids (BRs) are growth-promoting steroidal hormones. Despite the importance of BRs in plant biology, the signal that initiates BR biosynthesis remains unknown. Among the enzymes involved in BR biosynthesis in Arabidopsis (Arabidopsis thaliana), DWARF4 catalyzes the rate-determining step. Through both the histochemical analysis of DWF4pro:GUS plants and the direct measurement of endogenous BR content, we discovered that BR biosynthesis is stimulated by auxin. When DWF4pro:GUS was subjected to auxin dose-response tests and a time-course analysis, GUS activity started to increase at an auxin concentration of 10 nm, rising noticeably after 1 h of auxin treatment. In addition, the analysis of the DWF4pro:GUS line in BR- and auxin-mutant backgrounds revealed that the induction by auxin requires auxin-signaling pathways but not BRs, which implies that auxin signaling directly controls BR biosynthesis. Furthermore, chromatin immunoprecipitation assays confirmed that auxin inhibits the binding of the transcriptional repressor, BZR1, to the DWF4 promoter. A microarray analysis that was designed to examine the transcriptomes after treatment with auxin alone or auxin plus brassinazole (a BR biosynthetic inhibitor) revealed that genes previously characterized as being auxin responsive are not properly regulated when BR biosynthesis is disrupted by brassinazole. Therefore, our results support the idea that auxin regulates BR biosynthesis, and that auxin thus relies on synthesized BRs for some of its growth-promoting effects in Arabidopsis.