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Manipulating gene expression in the host genome with high precision is crucial for controlling cellular function and behavior. Here, we present a precise, non-invasive, and tunable strategy for controlling the expression of multiple endogenous genes both in vitro and in vivo, utilizing ultrasound as the stimulus. By engineering a hyper-efficient dCas12a and effector under a heat shock promoter, we demonstrate a system that can be inducibly activated through thermal energy produced by ultrasound absorption. This system allows versatile thermal induction of gene activation or base editing across cell types, including primary T cells, and enables multiplexed gene activation using a single guide RNA array. In mouse models, localized temperature elevation guided by high-intensity focused ultrasound effectively triggers reporter gene expression in implanted cells. Our work underscores the potential of ultrasound as a clinically viable approach to enhance cell and gene-based therapies via precision genome and epigenome engineering.

 

The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. The method, which we named CLIP (for 'CRISPR for long-fragment integration via pseudovirus'), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and 'cut sites' to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. CLIP offers a scalable and efficient method for manufacturing engineered primary cells. 

Machine learning and multiplexed screening elucidates enhancer networks regulating gene expression in health and disease states. 

Abstract The control of the in-plane domain evolution in ferroelectric thin films is not only critical to understanding ferroelectric phenomena but also to enabling functional device fabrication. However, in-plane polarized ferroelectric thin films typically exhibit complicated multi-domain states, not desirable for optoelectronic device performance. Here we report a strategy combining interfacial symmetry engineering and anisotropic strain to design single-domain, in-plane polarized ferroelectric BaTiO 3 thin films. Theoretical calculations predict the key role of the BaTiO 3 /PrScO 3 $${({{{{{\boldsymbol{110}}}}}})}_{{{{{{\bf{O}}}}}}}$$ ( 110 ) O substrate interfacial environment, where anisotropic strain, monoclinic distortions, and interfacial electrostatic potential stabilize a single-variant spontaneous polarization. A combination of scanning transmission electron microscopy, piezoresponse force microscopy, ferroelectric hysteresis loop measurements, and second harmonic generation measurements directly reveals the stabilization of the in-plane quasi-single-domain polarization state. This work offers design principles for engineering in-plane domains of ferroelectric oxide thin films, which is a prerequisite for high performance optoelectronic devices.