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Biology subjects

Hirosawa, M.

Publications and source records attributed to Hirosawa, M..

2 recordsLinked to original sources

Transient let-7/miR-98 reprograms primed pluripotency to induce naivehuman PSCs

Naive human pluripotent stem cells (hPSCs) are valuable for modeling early development and enabling regenerative applications, yet resetting from the primed state remains inefficient and mechanistically unclear. We identify an unexpected role for miR-98-5p, a let-7 family microRNA (miRNA) typically linked to differentiation, in promoting naive induction. Using a synthetic mRNA screen containing diverse miRNA-responsive elements, we uncovered miR-98-5p as a potent enhancer of resetting. Single-cell RNA sequencing and targeted perturbations show that miR-98-5p directly represses NR6A1 while transiently activating BMP2/4 signaling, triggering network transitions. These changes destabilize the primed gene-regulatory network and enable acquisition of a naive state. Leveraging this mechanism, we establish a feeder-free, miRNA-based, editing-free protocol that boosts resetting efficiency from <10% to [~]80%. To our knowledge, this is the first demonstration that a let-7 miRNA can drive naive induction by coupling direct target repression with activation of pro-naive signaling, suggesting a generalizable reprogramming principle of transient network destabilization.

developmental biology↗

Programmable mammalian translational modulators by CRISPR-associated proteins

The complexity of synthetic genetic circuits relies on repertories of biological circuitry with high orthogonality. Although post-transcriptional circuitry relying on RNA-binding proteins (RBPs) qualifies as a repertory, the limited pool of regulatory devices hinders network modularity and scalability. Here we propose CaRTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Genomic Engineering) to repurpose CRISPR-associated (Cas) proteins as translational modulators. We demonstrate that a set of Cas proteins are able to repress (OFF) or activate (ON) the translation of mRNAs that contain a Cas-binding RNA motif in the 5-UTR. We designed 81 different types of translation OFF and ON switches and verified their functional characteristics. Many of them functioned as efficient translational regulators and showed orthogonality in mammalian cells. By interconnecting these switches, we designed and built artificial circuits, including 60 translational AND gates. Moreover, we show that various CRISPR-related technologies, including anti-CRISPR and split-Cas9 platforms, can be repurposed to control translation. Our Cas-mediated translational regulation is compatible with transcriptional regulation by Cas proteins and increases the complexity of synthetic circuits with fewer elements. CaRTRIDGE builds protein-responsive mRNA switches more than ever and leads to the development of both Cas-mediated genome editing and translational regulation technologies.

synthetic biology↗