Focused ultrasound-mediated brain genome editing

Abstract

Gene editing in the brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches mainly rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain.

Keywords: CRISPR/Cas9; blood–brain barrier; focused ultrasound; gene delivery; genome editing.

Fig. 1.

FUS to enhance systemic AAV/CRISPR vector delivery to the brain. (A) Schematic overview of FUS-mediated BBB opening. (B) FUS systems used in this study and the MRI images showing the transient opening induced by FUS (arrowed). (C) Biodistribution of AAV9/SaCas9 vector (N = 5 for the FUS group and N = 4 for control, adult male C57BL/6). Mice received the vector in a dose of 2 × 10¹¹ GC/mouse, and the disposition was quantified at week 2 post-administration. (D) SaCas9 expression in the brain when mice given with different AAV9/SaCas9 vectors. Adult male C57BL/6 were intravenously given the vector in a dose of 2 × 10¹¹ GC/mouse (N = 3). (E) Representative RNA in situ hybridization images to confirm the SaCas9 expression in the FUS-targeted region. (F) Disposition of AAV9/SaCas9 vector and (G) Gene editing efficiency in different brain regions (two biological repeats). For E–G, adult male C57BL/6 were received an intravenous AAV9/SaCas9 dose of 10¹² GC/mouse, and the brain was dissected at week 3 post-administration.

Fig. 2.

Validation of FUS-mediated brain gene editing in reporter mouse models. (A) Representative 3D Lightsheet images showing the Cas9-activated TdTomato signals (red) in the brain. (B) Gene editing efficiency quantified by Lightsheet microscope. (C) Efficiency quantification by immunostaining. (D) Gene editing efficiency in different brain regions. Two sets of serial sections were used for immunostaining and quantification for each sex. (E) In-house quantification of edited neurons in ROIs in FUS-AAV9/SaCas9–treated Ai9 mice by immunostaining. (F) Independent quantification of neuron editing performed by the SCGE SATC and representative confocal images. (G) Quantification of TagRFP+ neurons in ROIs in FUS-AAV9/SaCas9–treated TLR2 reporter mice by immunostaining and the representative confocal images. (H) Neuron editing performance determined with the experimental correction. For B and C and E–H, data are presented in dots and circles for the results from female and male mice, respectively.