The biggest challenge in using CRISPR technologies,which limits their widespread application in medicine,is off-target effects.These effects could,in principle,be minimized by ensuring that CRISPR is activated primari...The biggest challenge in using CRISPR technologies,which limits their widespread application in medicine,is off-target effects.These effects could,in principle,be minimized by ensuring that CRISPR is activated primarily in the targeted cells,thereby reducing the likelihood of unintended genetic modifications in non-target tissues.Therefore,the development of a light activatable CRISPR approach to dynamically control gene activation in both space and time would be highly beneficial.A drawback is that the overwhelming majority of recently introduced light activatable CRISPR systems require UV or blue light exposure,severely limiting the penetration depth of light in tissue at which CRISPR can be activated,and,in the case of UV light,raising safety concerns.A small number of systems that activate CRISPR using longer wavelengths are hindered by either slow light activation or issues related to toxicity and biocompatibility of the proposed techniques in humans.To address this,we developed a split-Cas9/dCas9 system in which activation is achieved through a near-infrared photocleavable dimerization complex.This photoactivation method can be safely used in humans in vivo,easily adapted to different split-Cas9/dCas9 systems,and enables rapid,spatially precise light activation across various cell types.展开更多
基金supported by US National Science Foundation grants EFRI 1830878,CBET 1948722,CBET 2220273,and CBET 2325317US National Institutes of Health grants R01 EB025173,R01 CA228029,R01 CA293050,R01 GM143749,and R21 AG085089.
文摘The biggest challenge in using CRISPR technologies,which limits their widespread application in medicine,is off-target effects.These effects could,in principle,be minimized by ensuring that CRISPR is activated primarily in the targeted cells,thereby reducing the likelihood of unintended genetic modifications in non-target tissues.Therefore,the development of a light activatable CRISPR approach to dynamically control gene activation in both space and time would be highly beneficial.A drawback is that the overwhelming majority of recently introduced light activatable CRISPR systems require UV or blue light exposure,severely limiting the penetration depth of light in tissue at which CRISPR can be activated,and,in the case of UV light,raising safety concerns.A small number of systems that activate CRISPR using longer wavelengths are hindered by either slow light activation or issues related to toxicity and biocompatibility of the proposed techniques in humans.To address this,we developed a split-Cas9/dCas9 system in which activation is achieved through a near-infrared photocleavable dimerization complex.This photoactivation method can be safely used in humans in vivo,easily adapted to different split-Cas9/dCas9 systems,and enables rapid,spatially precise light activation across various cell types.
