Spatiotemporally confined red light-controlled gene delivery at single-cell resolution using adeno-associated viral vectors

Abstract

Methodologies for the controlled delivery of genetic information into target cells are of utmost importance for genetic engineering in both fundamental and applied research. However, available methods for efficient gene transfer into user-selected or even single cells suffer from low throughput, the need for complicated equipment, high invasiveness, or side effects by off-target viral uptake. Here, we engineer an adeno-associated viral (AAV) vector system that transfers genetic information into native target cells upon illumination with cell-compatible red light. This OptoAAV system allows adjustable and spatially resolved gene transfer down to single-cell resolution and is compatible with different cell lines and primary cells. Moreover, the sequential application of multiple OptoAAVs enables spatially resolved transduction with different transgenes. The approach presented is likely extendable to other classes of viral vectors and is expected to foster advances in basic and applied genetic research.

Red Light Optogenetics in Neuroscience

“…The system is composed of an AAV2 vector attached to PIF6 and an engineered antibody-mimicking DARPin attached to plant phytochrome B. The selectivity of the system is based on the specific cell surface protein binding of the used DARPin and the inactivation of the natural AAV viral tropism, that is, the ability of the virus to infect certain cell types better compared to others ( Hörner et al, 2021 ).…”

Spatially Defined Gene Delivery into Native Cells with the Red Light‐Controlled OptoAAV Technology

“…The OptoAAV technology allows the spatially resolved delivery of transgenes into native target cells down to single‐cell resolution by the illumination with low‐intensity red and far‐red light (Hörner et al., 2021). The system is based on an engineered AAV vector of serotype 2 (OptoAAV) and an adapter protein.…”

“…To externally control transgene delivery, light is the optimal stimulus as it can be easily applied with high spatiotemporal precision in a reversible manner. As existing technologies for light‐controlled viral gene delivery were hampered by the need for cytotoxic ultraviolet (UV) light (Goater et al., 2000; Ito et al., 2004; Pandori & Sano, 2000; Pandori et al., 2002; Ulrich‐Vinther et al., 2002; Wang et al., 2019), the required uptake of viral vectors to both target and off‐target cells before optical control (Bonsted et al., 2006; Bonsted, Hogset, Hoover, & Berg, 2005; Goater et al., 2000; Gomez, Gerhardt, Judd, Tabor, & Suh, 2016; Hagihara et al., 2020; Hogset et al., 2002; Ito et al., 2004; Tahara et al., 2019; Ulrich‐Vinther et al., 2002; Wang et al., 2019), or the requirement for genetic pre‐engineering of the target cells to express the photoreceptor (Gomez et al., 2016), we recently published the OptoAAV technology that overcomes these limitations (Hörner et al., 2021). The OptoAAV system allows the spatially resolved delivery of transgenes into native target cells upon illumination with low‐intensity red light and is discussed in more detail in the section Background Information.…”

“…no. P06-07100) High-purity DNA of plasmid pHelper(Cell Biolabs, High-purity DNA of plasmid pRCVP2koA (gift from H. Büning, Hannover Medical School;Münch et al, 2013) High-purity DNA of plasmid pMH303(Hörner et al, 2021) High-purity DNA of plasmid pCMVgfp (gift from D.Centrifuge with swing-out rotor for 50-ml conical centrifuge tubes (Eppendorf, Centrifuge 5804 with rotor S-4-72) Water bath at 37°C (PEQLAB, WB-4MS) Centrifuge for 15-and 50-ml conical centrifuge tubes (Eppendorf, Centrifuge 5804 with rotor FA-45-6-30) Ultrasonic water bath (Elma, Elmasonic S 80 H) 15-ml conical centrifuge tubes (Greiner Bio-One, cat. no.…”

The Red Edge: Bilin-Binding Photoreceptors as Optogenetic Tools and Fluorescence Reporters

“…The authors demonstrated sequential and spatially defined delivery of different transgenes into native mammalian cell lines and primary cells with single cell resolution. 217 4.1.3.1. Orthogonal Control of Cellular Functions: Combining Photoreceptors of Wavelengths.…”