Figure 2.

Knock-in-driven expression of therapeutic proteins in APOC3-expressing cells

(A) Strategy for genome editing-mediated knock-in of human F9 in the TS-APOC3 site. Combination of CjCas9 and TS-APOC3 with the HR donor or HITI donor results in the expression of hF9 by the targeted APOC3 genomic structure after splicing of the knocked-in sequence containing the splicing acceptor (SA) and F9 and the bovine growth hormone polyadenylation signal (pA). UHR, upstream homology arm; DHR, downstream homology arm. (B) Relative percentages of GFP-positive Hep G2 cells (%) after transfection with GFP-CMV, HITI-donor-only, and HITI (CjCas9 and donor) (n = 4–5). The HITI donor structure is same as in the strategy (A), except that GFP cDNA was used instead of F9 cDNA. Note that only HITI-treated cells showed sustained GFP expression over time. (C) Indel frequencies of cells after FACS analysis at 3 weeks after transfection (n = 4–5). (D and E) F9 mRNA expression (D) and indel frequency (E) in Hep G2 cells transfected with HR or HITI donor in the absence or presence of the plasmid-expressing CjCas9 and TS-APOC3 (n = 4). Ctrl: untransfected cells. Primer locations for quantitative RT-PCR designed within the F9 CDS (left) or at a junction between APOC3 exon 1 and the F9 CDS (right) are indicated by arrows (D). Data are presented as means ± SEM, and statistical analysis was performed using the Student t test. ∗∗p < 0.01.