Fig. 2 |. Gene editing in mouse lung with RCB-4-8 LNP-mRNA.

a, Measuring RCB-4-8 LNP-Cre-mRNA mediated editing using Lox-STOP-Lox tdTomato (Ai9) reporter mice. The Cre recombinase will delete the STOP cassettes and activate the tdTomato reporter. b, Ai9 mice were intratracheally administered with either one or three doses (over four days) of Cre-mRNA. To study the impact of AAV5 immunogenicity on the LNP, we delivered 6×10¹⁰ AAV5 with a dummy sequence in the mouse lung one week before dosing LNP-mRNA. Three days after the last dose, the lungs were collected and analyzed by flow cytometry (n=3 mice for each group). Results were obtained from three mice and presented as mean ± SD. c, Representative native fluorescence images of lung sections are shown. Results were obtained from 3 independent experiments. Saline serves as a negative control. Scale bar: 100μm. d, Paraffin-embedded lung sections were stained with antibodies for tdTomato. e, Quantification of IHC in d. Results were obtained from three mice (n=5 sections per mouse) and presented as mean ± SD. ***P<0.001 by one-way ANOVA with Tukey’s multiple comparisons test. f. Genomic DNA was collected from mice (n=3/group) treated with LNP-Cre mRNA or controls. PCR was performed with primers flanking the Ai9 locus. WT: wide type. M, marker. g, Quantification of tdTomato⁺ club cells. Results were obtained from three mice (3 airways per mouse) and presented as mean ± SD. h, Quantification of tdTomato⁺ ciliated cells. Results were obtained from three mice (3 airways per mouse) and presented as mean ± SD. i, Measuring RCB-4-8 LNP-SpCas9-mRNA/sgRNA mediated NHEJ using Ai9 reporter mice. The sgAi9 or sgA and sgB will delete the STOP cassettes and activate the tdTomato reporter. Ai9 mice were intratracheally administered with low or high doses of SpCas9-mRNA/sgAi9. To develop a combined viral and non-viral delivery of CRISPR system components in vivo, we intratracheally delivered 6×10¹⁰ scAAV5-sgA-sgB-GFP one week before dosing LNP-SpCas9-mRNA. Three days after the last dose, the lungs were collected. j-k, Quantification of tdTomato positive cells (k) and representative native fluorescence images of lung sections (j) are shown. LNP_high-SpCas9mRNA serves as a negative control. Scale bar: 100μm. n=9 sections from 3 mice. Error bars are S.D. Results were obtained from three mice (3 airways per mouse) and presented as mean ± SD.