RNA regulation in plants.

Bacterial small regulatory RNAs.

A cuffed CRISPR guide RNA for microRNA activity-dependent genome editing

Cells in multicellular eukaryotic systems are diverse biological units, with characteristics and functions determined by their molecular profiles. CRISPR–Cas9 genome editing has been widely used across biology to modulate gene expression and study gene function. However, there is currently no versatile and scalable method for editing a cell’s genome in response to endogenous cellular signals. Here, we report the engineering of a CRISPR guide RNA that efficiently confers genome editing in response to the catalytic activity of a target microRNA (miRNA) within a cell. miRNAs are short non-coding RNAs that are widely conserved across eukaryotes and can cleave their target RNA through almost perfect base pairing. In mammals, miRNAs are largely involved in development and homeostasis as well as disease progression and developmental disorders. To leverage these properties for genome editing, we developed a cuffed guide RNA (cgRNA) which is composed of a permutated order of sequence domains from the commonly used single guide RNA (sgRNA). These permutated domains were then concatenated with a miRNA target sequence, yielding a warped guide RNA that is inactive until cleaved by a complementary miRNA. We demonstrated that cgRNA enabled efficient miRNA activity-dependent genome editing in human and mouse cell lines. Biochemical and structural analyses revealed three stages of inhibition of the CRISPR genome-editing pathway for unprocessed cgRNA. Utilizing a lentiviral library of cgRNAs containing miRNA targets covering mouse genome-wide miRNAs, we identified miRNA cleavage activities and their sequence specificities in mouse embryonic stem cells and during smooth muscle cell differentiation. Furthermore, we showed that endogenous mRNA expression could be irreversibly recorded into a DNA sequence using a cgRNA targeted by a synthetic miRNA repeat. cgRNA is a simple, robust, miRNA activity-gated genome editing system that could facilitate the development of cell state-specific genome editing, the mapping of miRNA activity and gene expression landscapes, and the recording of molecularly determined cell states during the long-term progression of multicellular systems.

Research Progress on the Regulation of Plant Growth, Development and Stress Responses by Non-Coding RNAs

Design of Conditional Guide RNAs for the Logical Regulation of Gene Expression.

This chapter describes the design of cgRNAs and provides detailed protocols for their in vivo characterization in E. coli, and shows how cgRNAs can be integrated into endogenous gene circuits to achieve sophisticated and logical regulation of gene expression.

Functional genomics approaches for understanding the role of noncoding RNAS in human diseases

The latest functional genomics strategies used to examine NCRNAS are discussed and their transformative ability in accurate therapy is highlighted, highlighting their transformative ability in accurate therapy.

The expanding functional landscape of alternative splicing in plants.

Alternative splicing (AS) massively expands transcriptome diversity but does so within a densely interconnected regulatory network that challenges simple, linear models of gene expression. Despite its pervasiveness, principles governing AS choices and their functional outcomes are only beginning to emerge. Recent work in plants places AS at the interface of transcriptional dynamics, RNA fate, and protein output, raising fundamental questions about how transcript diversity translates into function. In parallel, splicing factors are increasingly recognized as multifunctional regulators with 'moonlighting' roles beyond pre-mRNA processing, including roles in chromatin, transcription, silencing, and stress responses. Here, we discuss how splicing-dependent and splicing-independent activities are integrated and propose a framework in which splicing factors act as regulatory hubs coordinating gene expression across multiple layers.