Engineering a far-red light–activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors

Abstract

<jats:p>It is widely understood that CRISPR-Cas9 technology is revolutionary, with well-recognized issues including the potential for off-target edits and the attendant need for spatiotemporal control of editing. Here, we describe a far-red light (FRL)–activated split-Cas9 (FAST) system that can robustly induce gene editing in both mammalian cells and mice. Through light-emitting diode–based FRL illumination, the FAST system can efficiently edit genes, including nonhomologous end joining and homology-directed repair, for multiple loci in human cells. Further, we show that FAST readily achieves FRL-induced editing of internal organs in tdTomato reporter mice. Finally, FAST was demonstrated to achieve FRL-triggered editing of the <jats:italic>PLK1</jats:italic> oncogene in a mouse xenograft tumor model. Beyond extending the spectrum of light energies in optogenetic toolbox for CRISPR-Cas9 technologies, this study demonstrates how FAST system can be deployed for programmable deep tissue gene editing in both biological and biomedical contexts toward high precision and spatial specificity.</jats:p>

A Blue Light-Inducible CRISPR-Cas9 System for Inhibiting Progression of Melanoma Cells

“…In fact, researchers have made many successful attempts in the treatment of diseases with light control devices. The research of AAV has entered the clinical treatment stage, the combination of AAV and light control devices is feasible (Wang E. et al, 2019;Yang et al, 2020;Yu et al, 2020).…”

Spatiotemporal control of CRISPR/Cas9 gene editing

“…In recent years, physical control has become an increasingly popular strategy in the spatiotemporal control of the CRISPR/Cas9 gene editing system due to its high spatiotemporal precision and noninvasiveness. 18 , 131 Generally, the main mechanism of the physical strategy is that some physically responsive elements, such as optical-responsive, heat-responsive, magnetic- and ultrasound-responsive components, are used to construct CRISPR platforms or delivery carriers (Figs. 6, 7 ).…”

A semi-synthetic red light photoswitch for optogenetic control of protein activity

“…One of the bacterial phytochromes, BphS, is activated by red light to convert guanylate triphosphate (GTP) into cyclic diguanylate monophosphate (cdi-GMP). BphS allows for red-light inducible transcription of a target gene by using it together with additional modules, such as the c-di-GMP-responsive hybrid transactivator p65-VP64-NLS-BldD, the chimeric promoter PFRLx and YhjH phosphodiesterase 22,23 . However, the BphSbased system requiring many components is complicated, does not enable for direct manipulation of protein activity, and could cause adverse effects on mammalian cells through an endogenous target of c-di-GMP.…”