An Essential Role for miRNA167 in Maternal Control of Embryonic and Seed Development.

Maternal cells play a critical role in ensuring the normal development of embryos, endosperms, and seeds. Mutations that disrupt the maternal control of embryogenesis and seed development are difficult to identify. Here, we completely deleted four MICRORNA167 (MIR167) genes in Arabidopsis (Arabidopsis thaliana) using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) genome-editing technology. We found that plants with a deletion of MIR167A phenocopied plants overexpressing miRNA167-resistant versions of Auxin Response Factor6 (ARF6) or ARF8, two miRNA167 targets. Both the mir167a mutant and the ARF overexpression lines were defective in anther dehiscence and ovule development. Serendipitously, we found that the mir167a (♀) × wild type (♂) crosses failed to produce normal embryos and endosperms, despite the findings that embryos with either mir167a+/- or mir167a-/- genotypes developed normally when mir167a+/- plants were self-pollinated, revealing a central role of MIR167A in maternal control of seed development. The mir167a phenotype is 100% penetrant, providing a great genetic tool for studying the roles of miRNAs and auxin in maternal control. Moreover, we found that mir167a mutants flowered significantly later than wild-type plants, a phenotype that was not observed in the ARF overexpression lines. We show that the reproductive defects of mir167a mutants were suppressed by a decrease of activities of ARF6, ARF8, or both. Our results clearly demonstrate that MIR167A is the predominant MIR167 member in regulating Arabidopsis reproduction and that MIR167A acts as a maternal gene that functions largely through ARF6 and ARF8.

Personalized circulating tumor DNA detection to monitor immunotherapy efficacy and predict outcome in locally advanced or metastatic non-small cell lung cancer.

OBJECTIVE: Immune checkpoint inhibitors (ICIs) or combined with chemotherapy exhibit substantial efficacy for the treatment of advanced non-small cell lung cancer (NSCLC). However, reliable biomarkers that can monitor response to first-line ICIs ± chemotherapy remain unclear. METHODS: A total of 16 tumor tissues and 46 matched peripheral blood samples at baseline and during treatment were retrospectively collected from 19 locally advanced or metastatic NSCLC patients. The circulating tumor DNA (ctDNA) burden by tumor-informed assay was detected to monitor and predict the therapeutic response and survival of NSCLC patients treated with first-line ICIs or plus chemotherapy. RESULTS: We found that ctDNA was only positively detected in one patient by tumor-agnostic assay with a mean variant allele fraction (VAF) of 6.40%, whereas it was positively detected in three patients by tumor-informed assay with a mean VAF of 8.83%, 0.154%, and 0.176%, respectively. Tumor-informed assays could sensitively detect ctDNA in 93.75% (15/16) of patients. Trends in the level of ctDNA from baseline to first evaluation was consistent with the radiographic changes. There was a greater decrease in ctDNA after treatment compared with baseline in patients with partial response compared to patients with stable disease/progressive disease. Patients with over a 50% reduction in ctDNA had a significant progression-free survival and overall survival benefit. CONCLUSION: The tumor-informed assay was favorable for ctDNA detection, and early dynamic changes in plasma ctDNA may be a valuable biomarker for monitoring the efficacy and predicting the outcome in advanced NSCLC patients treated with first-line ICIs ± chemotherapy.

Two homologous INDOLE-3-ACETAMIDE (IAM) HYDROLASE genes are required for the auxin effects of IAM in Arabidopsis.

Indole-3-acetamide (IAM) is the first confirmed auxin biosynthetic intermediate in some plant pathogenic bacteria. Exogenously applied IAM or production of IAM by overexpressing the bacterial iaaM gene in Arabidopsis causes auxin overproduction phenotypes. However, it is still inconclusive whether plants use IAM as a key precursor for auxin biosynthesis. Herein, we reported the isolation IAMHYDROLASE1 (IAMH1) gene in Arabidopsis from a forward genetic screen for IAM-insensitive mutants that display normal auxin sensitivities. IAMH1 has a close homolog named IAMH2 that is located right next to IAMH1 on chromosome IV in Arabidopsis. We generated iamh1 iamh2 double mutants using our CRISPR/Cas9 gene editing technology. We showed that disruption of the IAMH genes rendered Arabidopsis plants resistant to IAM treatments and also suppressed the iaaM overexpression phenotypes, suggesting that IAMH1 and IAMH2 are the main enzymes responsible for converting IAM into indole-3-acetic acid (IAA) in Arabidopsis. The iamh double mutants did not display obvious developmental defects, indicating that IAM does not play a major role in auxin biosynthesis under normal growth conditions. Our findings provide a solid foundation for clarifying the roles of IAM in auxin biosynthesis and plant development.

Fluorescence Marker-Assisted Isolation of Cas9-Free and CRISPR-Edited Arabidopsis Plants.

CRISPR/Cas9 gene editing technology has successfully introduced modifications at target DNA sequences in many plant species including Arabidopsis. After the target gene is edited, the CRISPR/Cas9 construct needs to be removed to ensure genetic stability and to gain any regulatory approval for commercial applications. However, removal of the transgenes by genetic segregation, backcross, and genotyping is very laborious and time-consuming. The methods we report here allow fast and effective isolation of transgene-free T2 Arabidopsis plants with the desired modifications at the target genes. We express a fluorescence protein mCherry under the control of a seed-specific promoter At2S3 and placed the cassette into the CRISPR/Cas9 vector. Therefore, we can use mCherry as a proxy for the presence of Cas9, and we are able to visually isolate the Cas9-free Arabidopsis plants with heritable mutations at the T2 generation. We targeted two sites in the ABP1 gene to demonstrate the effectiveness of our approach.