Fig. 3. Highly efficient multiplexed genome editing was achieved in vivo.

a Schematic illustration shows how delivery of Cas9/sgTOM RNPs activates Td-Tom expression in Td-Tomato transgenic mice. 5A2-DOT-X LNPs were injected into Td-Tom mice locally (via intra-muscle or intra-brain injections) and systemically (via IV injection through tail vein). In vivo imaging of Td-Tom mice after intra-muscle (1 mg kg⁻¹ sgTOM) (b) or intra-brain (0.15 mg kg⁻¹ sgTOM) (d) injection of 5A2-DOT-10 Cas9/sgTOM showed bright red fluorescence in the leg muscle or brain tissue (respectively). Successful CRISPR-Cas gene editing was further confirmed by confocal imaging of c muscle and e brain tissue sections. Scale bar: 20 μm. 5A2-DOT-10 enabled higher gene editing efficiency than positive control RNAiMAX, which has previously been used for local RNP injections. f In vivo imaging of Td-Tom mice after intravenous (IV) injection of 5A2-DOT-X Cas9/sgTOM LNPs with different molar percentages of DOTAP. Td-Tom fluorescence, as a downstream readout of DNA editing, showed that low DOTAP percentages facilitated liver editing while high DOTAP percentages facilitated lung editing (1.5 mg kg⁻¹ sgTOM, IV). g Successful CRISPR-Cas gene editing was further confirmed by confocal imaging. Scale bar: 20 μm. h The T7EI cleavage assay was performed on DNA isolated from liver and lung tissues after systemic IV treatment with 5A2-DOT-5, 5A2-DOT-10, 5A2-DOT-50, and 5A2-DOT-60 encapsulating Cas9/sgPTEN. Red arrows indicate cleavage bands generated. Indels (%) was calculated using next generation sequencing (NGS) of DNA isolated from harvested tissues. i 5A2-DOT-50 LNPs containing pooled sgRNAs for six targets (sgTOM, sgP53, sgPTEN, sgEml4, sgALK, and sgRB1) (5A2-DOT-50-Pool) were administered to td-Tom mice IV at total RNA dose of 2 mg kg⁻¹ (0.33 mg kg⁻¹ each sgRNA). Gene editing at the TOM locus was confirmed by in vivo imaging and j editing of the other five loci was confirmed using the T7EI assay on lung tissues. Indels percentages were measured using Sanger sequencing and TIDE analysis. Red arrows indicate cleavage bands generated. Data of c, e, g, h, and j were repeated three times independently with similar results.