The ability to precisely inactivate or modify genes in model organisms helps us understand the mysteries of life. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a revolutionary technology that could generate targeted mutants, has facilitated notable advances in plant science. Genome editing with CRISPR/Cas9 has gained great popularity and enabled several technical breakthroughs. Herein, we briefly introduce the CRISPR/Cas9, with a focus on the latest breakthroughs in precise genome editing (e.g., base editing and prime editing), and we summarize various platforms that developed to increase the editing efficiency, expand the targeting scope, and improve the specificity of base editing in plants. In addition, we emphasize the recent applications of these technologies to plants. Finally, we predict that CRISPR/Cas9 and CRISPR/Cas9-based genome editing will continue to revolutionize plant science and provide technical support for sustainable agricultural development.

DNA sequencing is vital for many aspects of biological research and diagnostics. Despite the development of second and third generation sequencing technologies, Sanger sequencing has long been the only choice when required to precisely track each sequenced plasmids or DNA fragments. Here, we report a complete set of novel barcoding and assembling system, Highly-parallel Indexed Tagmentation-reads Assembled Consensus sequencing (HITAC-seq), that could massively sequence and track the identities of each individual sequencing sample. With the cost of much less than that of single read of Sanger sequencing, HITAC-seq can generate high-quality contiguous sequences of up to 10 kilobases or longer. The capability of HITAC-seq was confirmed through large-scale sequencing of thousands of plasmid clones and hundreds of amplicon fragments using approximately 100 pg of input DNAs. Due to its long synthetic length, HITAC-seq was effective in detecting relatively large structural variations, as demonstrated by the identification of a ∼1.3 kb Copia retrotransposon insertion in the upstream of a likely maize domestication gene. Besides being a practical alternative to traditional Sanger sequencing, HITAC-seq is suitable for many high-throughput sequencing and genotyping applications.

Caste differentiation in social hymenopterans is an intriguing example of phenotypic plasticity. However, the co-ordination among gene regulatory factors to mediate caste differentiation remains inconclusive. In this study, we determined the role of gene regulation and related epigenetic processes in pre-imaginal caste differentiation in the primitively eusocial bumblebee Bombus terrestris. By combining RNA-Seq data from Illumina and PacBio and accurately quantifying methylation at whole-genomic base pair resolution, we found that queens, workers, and drones mainly differentiate in gene expression but not in alternative splicing and DNA methylation. Gynes are the most distinct with the lowest global level of whole-genomic methylation and with the largest number of caste-specific transcripts and alternative splicing events. By contrast, workers exhibit few uniquely expressed or alternatively spliced genes. Moreover, several genes involved in hormone and neurotransmitter metabolism are related to caste differentiation, whereas several neuropeptides are linked with sex differentiation. Despite little genome-wide association among differential gene expression, splicing, and differential DNA methylation, the overlapped gene ontology (GO) terms point to nutrition-related activity. Therefore, variations in gene regulation correlate with the behavioral differences among castes and highlight the specialization of toolkit genes in bumblebee gynes at the beginning of the adult stage.

Coordination between the sporophytic tissue and the gametic pollen within anthers is tightly controlled to achieve the optimal pollen fitness. Glucose-6-phosphate/phosphate translocator (GPT) transports glucose-6-phosphate, a key precursor of starch and/or fatty acid biosynthesis, into plastids. Here, we report the functional characterization of OsGPT1 in the rice anther development and pollen fertility. Pollen grains from homozygous osgpt1 mutant plants fail to accumulate starch granules, resulting in pollen sterility. Genetic analyses reveal a sporophytic effect for this mutation. OsGPT1 is highly expressed in the tapetal layer of rice anther. Degeneration of the tapetum, an important process to provide cellular contents to support pollen development, is impeded in osgpt1 plants. In addition, defective intine and exine are observed in the pollen from osgpt1 plants. Expression levels of multiple genes that are important to tapetum degeneration or pollen wall formation are significantly decreased in osgpt1 anthers. Previously, we reported that AtGPT1 plays a gametic function in the accumulation of lipid bodies in Arabidopsis pollen. This report highlights a sporophytic role of OsGPT1 in the tapetum degeneration and pollen development. The divergent functions of OsGPT1 and AtGPT1 in pollen development might be a result of their independent evolution after monocots and dicots diverged.

Juvenile hormone (JH) is one of the most important hormones in insects since it is essential for insect development. The mechanism by which JH affects the central nervous system still remains a mystery. In this study, we demonstrate that one of the JH receptors, Methoprene-tolerant (Met), is important for the control of neurite development and sleep behavior in Drosophila. With the identification of Met-expressing glial cells, the mechanism that Met negatively controls the mushroom body (MB) β lobes fusion and positively maintains pigment-dispersing factor sLNvs projection pruning has been established. Furthermore, despite the developmental effects, Met can also maintain nighttime sleep in a development-independent manner through the α/β lobe of MB. Combining analyses of neuronal morphology and entomological behavior, this study advances our understanding of how the JH receptor regulates the nervous system.

The vaginal microbiota is less complex than the gut microbiota, and the colonization of Lactobacillus in the female vagina is considered to be critical for reproductive health. Oral probiotics have been suggested as promising means to modulate vaginal homeostasis in the general population. In this study, 60 Chinese women were followed for over a year before, during, and after treatment with the probiotics Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14. Shotgun metagenomic data of 1334 samples from multiple body sites did not support a colonization route of the probiotics from the oral cavity to the intestinal tract and then to the vagina. Our analyses enable the classification of the cervicovaginal microbiome into a stable state and a state of dysbiosis. The microbiome in the stable group steadily maintained a relatively high abundance of Lactobacilli over one year, which was not affected by probiotic intake, whereas in the dysbiosis group, the microbiota was more diverse and changed markedly over time. Data from a subset of the dysbiosis group suggests this subgroup possibly benefited from supplementation with the probiotics, indicating that probiotics supplementation can be prescribed for women in a subclinical microbiome setting of dysbiosis, providing opportunities for targeted and personalized microbiome reconstitution.

Mitochondrial diseases are caused by variants in both mitochondrial and nuclear genomes. A nuclear gene HPDL (4-hydroxyphenylpyruvate dioxygenase-like), which encodes an intermembrane mitochondrial protein, has been recently implicated in causing a neurodegenerative disease characterized by pediatric-onset spastic movement phenotypes. Here, we report six Chinese patients with bi-allelic HPDL pathogenic variants from four unrelated families showing neuropathic symptoms of variable severity, including developmental delay/intellectual disability, spasm, and hypertonia. Seven different pathogenic variants are identified, of which five are novel. Both fibroblasts and immortalized lymphocytes derived from patients show impaired mitochondrial respiratory function, which is also observed in HPDL-knockdown (KD) HeLa cells. In these HeLa cells, overexpression of a wild-type HPDL gene can rescue the respiratory phenotype of oxygen consumption rate. In addition, a decreased activity of the oxidative phosphorylation (OXPHOS) complex Ⅱ is observed in patient-derived lymphocytes and HPDL-KD HeLa cells, further supporting an essential role of HPDL in the mitochondrial respiratory chain. Collectively, our data expand the clinical and mutational spectra of this mitochondrial neuropathy and further delineate the possible disease mechanism involving the impairment of the OXPHOS complex Ⅱ activity due to the bi-allelic inactivations of HPDL.

Donkey (Equus asinus) is an important livestock animal in China because of its draft and medicinal value. After a long period of natural and artificial selection, the variety and phenotype of donkeys have become abundant. We clarified the genetic and demographic characteristics of Chinese domestic donkeys and the selection pressures by analyzing 78 whole genomes from 12 breeds. According to population structure, most Chinese domestic donkeys showed a dominant ancestral type. However, the Chinese donkeys still represented a significant geographical distribution trend. In the selective sweep, gene annotation, functional enrichment, and differential expression analyses between large and small donkey groups, we identified selective signals, including NCAPG and LCORL, which are related to rapid growth and large body size. Our findings elucidate the evolutionary history and formation of different donkey breeds and provide theoretical insights into the genetic mechanism underlying breed characteristics and molecular breeding programs of donkey clades.