Fig. 5: SpyCas9-mSA RNP delivery enables off-target editing discovery in the lung.

a Delivery of RNP and donor DNA in vitro. Mouse N2A cells were transfected by Lipofectamine CRISPRmax with SpyCas9-mSA* sgActin (UTR) RNP and IRES-GFP donor (dsDNA or biotin-dsDNA). Flow cytometry analysis for GFP⁺ cells was performed 4 days after transfection. Results from three independent experiments are presented as mean ± SEM. ***P < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. b For GUIDE-tag analysis in lung, Ai9 mice were injected intratracheally with polymer-stabilized sgAi9-SpyCas9-mSA* RNP, sgPcsk9 SpyCas9-mSA* RNP plus biotin-iGUIDE donor (n = 2). GUIDE-tag libraries were prepared with gDNA isolated from sorted tdTomato⁺ lung cells. c List of validated OT sites in the lung. Average UMI numbers and average indel frequencies determined by targeted amplicon sequencing of sorted Tomato+ cells are shown (ranked by indel%, n = 2 mice). Mismatches relative to the on-target site are shown with colored boxes. d Venn diagram comparing potential OT sites discovered in the lung and liver by GUIDE-tag. e Scatter plots of average indel frequency and average UMI% for validated sgPcsk9 off-target sites in the lung. Dashed lines represent the linear regression fit (Spearman’s correlation calculated). The p-value for Pearson’s correlation coefficient was determined by the two-tailed t-distribution table. Source data are provided as a Source Data file.