A versatile and robust cell purification system with an RNA-only circuit composed of microRNA-responsive ON and OFF switches

２つの合成mRNAスイッチを活用した純度の高い細胞選別システムの開発. 京都大学プレスリリース. 2022-01-06.Synthetic gene circuits that improve stem cell quality. 京都大学プレスリリース. 2022-01-06.Human induced pluripotent stem cells (iPSCs) are promising cell resources for cell therapy and drug discovery. However, iPSC-derived differentiated cells are often heterogenous and need purification using a flow cytometer, which has high cost and time consumption for large-scale purification. MicroRNAs (miRNAs) can be used as cell selection markers, because their activity differs between cell types. Here, we show miRNA-responsive ON and OFF switch mRNAs for robust cell purification. The ON switch contains a miRNA-target sequence after the polyadenylate tail, triggering translational activation by sensing the target miRNA. By designing RNA-only circuits with miRNA-ON and -OFF switch mRNAs that encode a lethal ribonuclease, Barnase, and its inhibitor, Barstar, we efficiently purified specific cell types, including human iPSCs and differentiated cardiomyocytes, without flow cytometry. Synthetic mRNA circuits composed of ON and OFF switches provide a safe, versatile, and time-saving method to purify various cell types for biological and clinical applications

Programmable mammalian translational modulators by CRISPR-associated proteins

mRNAスイッチを用いた哺乳類細胞内コンピューティングの基盤構築. 京都大学プレスリリース. 2023-04-20.Establishing the basis for mammalian intracellular computing with mRNA switches. 京都大学プレスリリース. 2023-04-21.Translational modulation based on RNA-binding proteins can be used to construct artificial gene circuits, but RNA-binding proteins capable of regulating translation efficiently and orthogonally remain scarce. Here we report CARTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Gene control) to repurpose Cas proteins as translational modulators in mammalian cells. We demonstrate that a set of Cas proteins efficiently and orthogonally repress or activate the translation of designed mRNAs that contain a Cas-binding RNA motif in the 5’-UTR. By linking multiple Cas-mediated translational modulators, we designed and built artificial circuits like logic gates, cascades, and half-subtractor circuits. Moreover, we show that various CRISPR-related technologies like anti-CRISPR and split-Cas9 platforms could be similarly repurposed to control translation. Coupling Cas-mediated translational and transcriptional regulation enhanced the complexity of synthetic circuits built by only introducing a few additional elements. Collectively, CARTRIDGE has enormous potential as a versatile molecular toolkit for mammalian synthetic biology

マイクロRNA応答性CRISPR-Cas9スイッチを用いた細胞種特異的なゲノム編集

京都大学0048新制・課程博士博士(医科学)甲第21689号医科博第93号新制||医||1036(附属図書館)京都大学大学院医学研究科医科学専攻(主査)教授 斎藤 通紀, 教授 中川 一路, 教授 竹内 理学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch.

細胞種に応じてゲノム編集を制御する技術を開発. 京都大学プレスリリース. 2017-05-22.The CRISPR-Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR-Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information