Design guide for synthetic small regulatory RNAs for high-efficiency gene knockdown in bacteria.

A portable Cas6f-based system for multiplex translational repression in bacteria

Engineered small RNAs (sRNAs) enable programmable gene knockdowns and support metabolic engineering and multiplex regulation in model bacteria. Still, precise, tunable, and multiplex gene repression remains a challenge in synthetic biology. Common tools can impose genetic burden, depend on host RNA factors, or do not transfer well across species. Here we present MORTISE (Multiplex, ORthogonal Translation Interference SystEm), a compact Cas6f-based platform for programmable translational repression in Gram-negative bacteria. The system functions without host Hfq or RNases and operates robustly in Escherichia coli and Pseudomonas putida. We demonstrate repression in both species using chromosomal reporter assays, with performance improving when guide and target transcription are matched and when the translation initiation region is targeted. Single-promoter multiplexing enables simultaneous knockdowns and a cloning toolbox facilitates assembly of up to nine guides in a single step. Finally, MORTISE is leveraged to boost malonyl-coenzyme A–dependent production in P. putida, supporting pathway balancing. Engineered small RNAs (sRNAs) enable programmable gene knockdowns and support metabolic engineering and multiplex regulation in model bacteria. Here the authors introduce MORTISE, a compact Cas6f-based RNA system to repress target bacterial genes without relying on host RNA factors, enables multiplex control across bacteria, and supports pathway balancing when genetic knockouts are unsuitable.

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.

Bacterial small regulatory RNAs.

A decoupled transcription platform enables tunable and predictable gene expression in yeast

Predictable control of gene expression is essential for building genetic circuits and improving metabolic pathways, but conventional promoter libraries often behave unpredictably when genes are combined. Here we develop CRISPR-Activated Promoter-based Orthogonal expression (CAPO), a quantitative platform for controlling multiple genes in yeast. CAPO uses synthetic CRISPR-activated promoters that remain silent until matching guide RNAs recruit dCas9-VPR. We tune each gene by varying guide RNA abundance with defined T7 promoters, while keeping regulatory channels orthogonal. CAPO reaches expression levels comparable to strong native yeast promoters, maintains low background activity, and preserves promoter-strength order across different genes. We apply CAPO to program broad fluorescence color outputs and to rapidly optimize lycopene and 3-hydroxypropionic acid biosynthesis. These results establish CAPO as a scalable platform for predictable engineering of eukaryotic gene networks. Efficient bioproduction using eukaryotes, such as engineered Saccharomyces cerevisiae, requires precise control over gene expression. Here, authors develop CAPO, a CRISPR-guided system that tunes gene activity in yeast and enables multiplex colour generation and faster optimization of metabolic pathways.

Construction of a Tl-CRISPRi Genetic Circuit in Bacteria for Translation-Level Gene Knockdown.

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.