Fig 1: Improved targeting of the Apoa1 locus in neonatal mice injected at P4(A) Experimental design and AAV-Donor scheme. C57BL/6J mice were subcutaneously injected with AAV-CRISPR (5 × 1011 GC) and/or an AAV-Donor template (5 × 1011 GC) encoding a far-red fluorescent protein (mKate2) at P4 and liver and plasma were evaluated for the expression of the mKate2 protein at 20 weeks post-injection. Control mice were injected with AAV-GFP (5 × 1011 GC). (B) Integration PCR on liver showed two main products corresponding to correct HDR (1,139 bp) and NHEJ insertion (2,031 bp) of AAV-Donor at the Apoa1 locus. Minus (–) indicates water-only control. (C) Frequency of Apoa1 alleles with NHEJ insertions of AAV genomes by ddPCR. (D) Frequency of correct HDR targeting of AAV-Donor by ddPCR. (E) Indel formation in the 3' UTR of Apoa1 by ICE analysis. (F) Representative immunohistochemistry for mKate2-FLAG (brown cells) in Apoa1-targeted mice. Scale bar is 100 µm. (G) Quantification of FLAG-positive hepatocytes relative to total nuclei per field. (H) Western blot of mKate2-FLAG in liver lysates with ß-tub as a loading control. (I and J) Western blot analysis of 2A-tagged (I) and total apoA1 (J) in plasma with aat as loading control. Aat shows a slight difference in protein expression based on sex: lanes 1, 4, 7, 8, 9, 10, 13, 14, and 15 are male mice; lanes 2, 3, 5, 6, 11, 12, and 16 are female mice. Four representative mice per group are shown in western blots. (K) Densitometry analysis of mKate2-FLAG in liver lysates relative to ß-tub loading control. Densitometry analysis of apoA1-2A (L) and apoA1 (M) in plasma relative to aat loading control. Data are shown as mean ± standard deviation (n = 5; n = 4 in densitometry analyses), with significance determined by one-way ANOVA followed by Tukey test. ***p < 0.001, ****p < 0.0001. (A) Created with BioRender.