Prime editing events revealed by next-generation sequencing (NGS). (D) Quantification of prime editing frequencies by PPE3b-V01 at five target sites. (E) Editing events revealed by NGS reads. (F) Schematics of the expression vectors of Plant Prime Editor 3 or 2-Version 2 (PPE3/2-V02). (G-I) Comparison of multiple PBS-RT pairs of different lengths for directing TCA insertion at the OsPDS target site, directing C to A base change at the OsDEP1 target site and directing TGA insertion at the OsDEP1 target site, respectively, by PPE3-V02. (J and K) Validation of prime editing outcomes by NGS at the OsPDS-pegR15, OsDEP1-pegR03, and OsDEP1-pegR10 target sites. (M) Comparison of PPE3-V02 and PPE2-V02 for precise editing at five target sites. (N) PPE2-V02 based prime editing events revealed by NGS for the OsPDS-sgRNA01 3T ins construct (for insertion of a T 3 nt downstream of the PBS). (O) PPE2-V02 based prime editing at another site with multiple PBS-RT pairs of different lengths. The experiments were done in rice protoplasts.Three biological replicates were used to assess the PPE3-V01 system (B-E), and two biological replicates were used to assess PPE3-V02 and PPE2-V02 systems (G-O). Error bars represent standard deviations of the biological replicates. For NGS-based genotyping data presentations (C, E, J, K, L, and N), the sequences (from top to bottom) include the wild-type (WT) sequence (protospacer underlined and PAM in bold), the expected prime editing outcome (Reference), confirmed precise prime editing events matching the expected prime editing outcome (PE_Ref), precise prime editing plus additional single nucleotide polymorphisms (e.g., PE_h01; h stands for haplotype) and deletions resulted from the NHEJ repair. The prime edited DNA nucleotides are highlighted in red.
Cryo-electron microscopy (cryo-EM) has emerged as an unprecedented tool to resolve protein structures at atomic resolution. Structural insights of biological samples not accessible by conventional X-ray crystallography and NMR can be explored with cryo-EM because measurements are carried out under near-native crystal-free conditions, and large protein complexes with conformational and compositional heterogeneity are readily resolved. RNA has remained underexplored in cryo-EM, despite its essential role in various biological processes. This review highlights current challenges and recent progress in using cryo-EM single-particle analysis to determine protein-free RNA structures, enabled by improvement in sample preparation and integration of multiple structural and biochemical methods.
Summary
MicroRNA168 (MIR168) is a key miRNA that targets the main RNA‐induced silencing complex component Argonaute 1 (AGO1) to regulate plant growth and environmental stress responses. However, the regulatory functions of MIR168 need to be further elucidated in rice. In this paper, we generated clean OsMIR168a deletion mutants by CRISPR‐Cas9 strategy. We then phenotypically and molecularly characterized these mutants. The rice OsMIR168a mutants grew rapidly at the seedling stage, produced more tillers and matured early. Compared to the wild‐type plants, the mutants were shorter at maturity and produced smaller spikelets and seeds. Analysis of gene expression showed that the transcription levels of OsMIR168a’s target genes such as OsAGO1a, OsAGO1b and OsAGO1d were elevated significantly in the OsMIR168a mutants. Intriguingly, OsAGO18, a member of a new AGO clade that is conserved in monocots, was confirmed to be a target of OsMIR168a not only by informatic prediction but also by expression analysis and a cell‐based cleavage assay in the OsMIR168a mutants. Many protein‐coding genes and miRNAs showed differential expression in the OsMIR168a mutants, suggesting OsMIR168a exerts a major transcriptional regulatory role, likely through its potential target genes such as OsAGO1s and OsAGO18. KEGG enrichment analysis of these differentially expressed genes pointed to OsMIR168a’s involvement in important processes such as plant hormone signalling transduction and plant–pathogen interaction. These data collectively support that the complex regulation module of OsMIR168a‐OsAGO1/OsAGO18‐miRNAs‐target genes contributes to agronomically important traits, which sheds light on miRNA‐mediated crop breeding.
The EORTC IN-PATSAT32 appears to be a reliable, valid, and acceptable instrument to use on cancer patients and is appropriate for measuring the patient satisfaction of Chinese patients.
Background: To quantitatively evaluate the possible effects of phacoemulsification cataract surgery on macular hemodynamics using optical coherence tomography angiography (OCTA). Methods Prospective observational study. Superficial and deep macular vascular densities as well as parameters of foveal avascular zone (FAZ) were measured preoperatively (baseline) and at 1 day, 1week and 4 weeks postoperation in normal eyes (≥22 mm and≤24 mm) of patients scheduled for phacoemulsification cataract surgery with intraocular lens implantation. Correlations between the rate of change of pre-and post-operative vascular densities and surgical parameters were analyzed.Results : 123 eyes of 107 patients were recruited. Compared with baseline measurements, no statistically significant variation was found in macular vascular densities of day one after surgery (P>0.05). While both superficial and deep macular vascular densities were significantly increased postoperatively on week 1 and week 4 (P<0.05; P<0.05). There were no statistically significant differences in any of the FAZ parameters between the baseline measurements and the entire followup period (P>0.05 for all). There were no statistically significant correlations between main surgical parameters and the macular vascular densities. Conclusions : In normal eyes, macular blood perfusion gradually increased after phacoemulsification cataract surgery and was stabilized in one week. Foveal avascular zone was basically stable before and after surgery. Main parameters and intraoperative perfusion of phacoemulsification surgery may not be the key factors affecting macular hemodynamics.
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