Obtaining the best igRNAs for bystander-less correction of all ABE-reversible pathogenic SNVs using high-throughput screening.

Imperfect -gRNA (igRNA) provides a simple strategy for single-base editing of a base editor. However, a significant number of igRNAs need to be generated and tested for each target locus to achieve efficient single-base reversion of pathogenic single nucleotide variations (SNVs), which hinders the direct application of this technology. To provide ready-to-use igRNAs for single-base and bystander-less correction of all the adenine base editor (ABE)-reversible pathogenic SNVs, we employed a high-throughput method to edit all 5,253 known ABE-reversible pathogenic SNVs, each with multiple systematically designed igRNAs, and two libraries of 96,000 igRNAs were tested. A total of 1,988 SNV loci could be single-base reversed by igRNA with a >30% efficiency. Among these 1,988 loci, 378 SNV loci exhibited an efficiency of more than 90%. At the same time, the bystander editing efficiency of 76.62% of the SNV loci was reduced to 0%, while remaining below 1% for another 18.93% of the loci. These ready-to-use igRNAs provided the best solutions for a substantial portion of the 4,657 pathogenic/likely pathogenic SNVs. In this work, we overcame one of the most significant obstacles of base editors and provide a ready-to-use platform for the genetic treatment of diseases caused by ABE-reversible SNVs.

First report of rootstalk rot of Hibiscus mutabilis caused by Fusarium oxysporum in China.

H. Mutabilis (Cotton rose or confederate rose) is a deciduous shrub in the Malvaceae family, with ornamental, medicinal and edible values (Fan et al. 2015). In May to August 2020, 40.4% of potted plants of H. mutabilis were found to have root and stalk rot in Chengdu Botanical Garden (E104°7'28″, N30°45'57″). At first the leaves of affected H. mutabilis turned yellow and wilted, followed by the stem and root cortex became dark brown and rotten. Finally, the whole plant died within two months. Root and stem produced white mycelium when the humidity exceeded 90%. Samples taken from the lesions were surface disinfested for 3 min in 4% sodium hypochlorite, rinsed in sterile water and plated on potato dextrose agar (PDA), 35 single-spore cultures with similar morphology isolated from symptomatic tissues were obtained and subcultured. After seven days at 25°C in the dark, the mycelium of a representative culture MFR1 covered the entire plate surface (9 cm diameter). The aerial mycelium of cultures were white and fluffy at first and produced lavender pigment on the back of the cultures in the later stage. After seven days, the cultures produced abundant sickle-shaped macroconidia which have 3 to 5 septations and some oval or oblong microconidia which have 0 to 1 septation. Macroconidia 22.35~46.67 µm (mean 32.11 µm) in length and 4.32~7.72 µm (mean 5.21 µm) in width (n = 100). Microconidia 7.10~21.85 µm (mean 11.62 µm) in length and 2.76~6.84 µm (mean 4.20 µm) in width (n = 100). Based on these characteristics, isolates were tentatively identified as Fusarium sp. (Crous et al. 2021). Pathogenicity was tested on 1-year-old potted seedlings of H. mutabilis by root-zone irrigation inoculation in Sichuan Agricultural University (Jia et al.2019). Conidia suspension (1×107conidia/mL,collected from MFR1 )was poured into the soil along the plant roots. The same amount of distilled water was poured around the roots of the control plants. All inoculated and control plants were incubated in the greenhouse (about 25 ± 2°C). The experiment was performed three times. Within 25 days after inoculation, all plants inoculated with pathogens showed symptoms similar to those in the field, whereas the controls remained symptomless. The pathogen was reisolated from all inoculated plants, and the cultural and morphological characteristics were the same as those of the original isolate. After DNA extraction and PCR amplification, the translation elongation factor 1-alpha (TEF) and RNA polymerase II second largest subunit (RPB2) genes of a representative culture MFR1, were sequenced (O'Donnell et al. 2010) and deposited in GenBank (accession numbers OK334295 and ON316728, respectively). The TEF and RPB2 sequences were 99.7% and 99.39% identical to those of F. oxysporum (MN892354 and MZ198892). The result was confirmed by multilocus phylogenetic analysis. Through morphological identification and molecular analyses, the pathogen was identified as F. oxysporum. F. oxysporum is known to infect cotton (Dowd et al.2004), soybean (Ellis et al.2016) and banana (Fourie et al.2011) among other hosts, but it is the first report of F. oxysporum infecting H. mutabilis in China or worldwide. This disease seriously reduced the survival rate of H. mutabilis and may become an important reason to hinder the increase of H. mutabilis in potted seedlings stage. Moreover, the findings will provide theoretical basis to solve the bottleneck problem affecting the popularization and propagation of H. mutabilis.

Identification and Characterization of Hibiscus mutabilis Varieties Resistant to Bemisia tabaci and Their Resistance Mechanisms.

Hibiscus mutabilis, the city flower of Chengdu, is culturally significant and has nutritional and medicinal benefits. However, frequent infestations of Bemisia tabaci have caused economic losses. This study aimed to identify insect-resistant H. mutabilis varieties. Over two years, varieties like Jinqiusong, Zuiyun, and Zuifurong showed moderate to high resistance based on reproductive indices. Assessments of antixenosis and developmental impacts revealed that adult B. tabaci exhibited low selectivity toward these resistant varieties, indicating a strong repellent effect. Gas chromatography-mass spectrometry analysis identified volatile organic compounds, such as alcohols, alkanes, and terpenes. Notably, 2-ethylhexanol and 6-methylheptanol exhibited repellent properties. Using nontargeted metabolomics, this study compared the metabolite profiles of the insect-resistant variety Jinqiusong (JQS), moderately resistant Bairihuacai (BRHC), and highly susceptible Chongbanbai (CBB) post B. tabaci infestation. Fifteen key metabolites were linked to resistance, emphasizing the phenylpropanoid biosynthesis pathway as crucial in defense. These findings offer a theoretical foundation for breeding insect-resistant H. mutabilis varieties and developing eco-friendly strategies against B. tabaci infestations.

Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells.

Base editors show promise for treating human genetic diseases, but most current systems use deaminases, which cause off-target effects and are limited in editing type. In this study, we constructed deaminase-free base editors for cytosine (DAF-CBE) and thymine (DAF-TBE), which contain only a cytosine-DNA or a thymine-DNA glycosylase (CDG/TDG) variant, respectively, tethered to a Cas9 nickase. Multiple rounds of mutagenesis by directed evolution in Escherichia coli generated two variants with enhanced base-converting activity-CDG-nCas9 and TDG-nCas9-with efficiencies of up to 58.7% for C-to-A and 54.3% for T-to-A. DAF-BEs achieve C-to-G/T-to-G editing in mammalian cells with minimal Cas9-dependent and Cas9-independent off-target effects as well as minimal RNA off-target effects. Additional engineering resulted in DAF-CBE2/DAF-TBE2, which exhibit altered editing windows from the 5' end to the middle of the protospacer and increased C-to-G/T-to-G editing efficiency of 3.5-fold and 1.2-fold, respectively. Compared to prime editing or CGBEs, DAF-BEs expand conversion types of base editors with similar efficiencies, smaller sizes and lower off-target effects.

A High-Quality, Chromosome-Level Genome Provides Insights Into Determinate Flowering Time and Color of Cotton Rose (Hibiscus mutabilis).

Hibiscus mutabilis (cotton rose) is a deciduous shrub or small tree of the Malvaceae family. Here, we report a chromosome-scale assembly of the H. mutabilis genome based on a combination of single-molecule sequencing and Hi-C technology. We obtained an optimized assembly of 2.68 Gb with a scaffold N50 length of 54.7 Mb. An integrated strategy of homology-based, de novo, and transcriptome-based gene predictions identified 118,222 protein-coding genes. Repetitive DNA sequences made up 58.55% of the genome, and LTR retrotransposons were the most common repetitive sequence type, accounting for 53.15% of the genome. Through the use of Hi-C data, we constructed a chromosome-scale assembly in which Nanopore scaffolds were assembled into 46 pseudomolecule sequences. We identified important genes involved in anthocyanin biosynthesis and documented copy number variation in floral regulators. Phylogenetic analysis indicated that H. mutabilis was closely related to H. syriacus, from which it diverged approximately 15.3 million years ago. The availability of cotton rose genome data increases our understanding of the species' genetic evolution and will support further biological research and breeding in cotton rose, as well as other Malvaceae species.

Automated high-throughput genome editing platform with an AI learning in situ prediction model.

A great number of cell disease models with pathogenic SNVs are needed for the development of genome editing based therapeutics or broadly basic scientific research. However, the generation of traditional cell disease models is heavily dependent on large-scale manual operations, which is not only time-consuming, but also costly and error-prone. In this study, we devise an automated high-throughput platform, through which thousands of samples are automatically edited within a week, providing edited cells with high efficiency. Based on the large in situ genome editing data obtained by the automatic high-throughput platform, we develop a Chromatin Accessibility Enabled Learning Model (CAELM) to predict the performance of cytosine base editors (CBEs), both chromatin accessibility and the context-sequence are utilized to build the model, which accurately predicts the result of in situ base editing. This work is expected to accelerate the development of BE-based genetic therapies.