Engineering of a human genomic HLF reporter. (A) Outline of the HLF reporter–targeting strategy using CRISPR/Cas9 and rAAV6. A site-specific double-strand (ds) break at the HLF stop codon (orange) located in exon 4 is generated by a Cas9/sgHLF RNP complex. This stimulates HR with a single-stranded donor template delivered through rAAV6 infection. The resulting HR event results in a transgenic locus that coexpresses the HLF open reading frame and a multifunctional ZsGreen (ZsG) expression cassette connected the endogenous HLF open reading frame by an encephalomyocarditis virus internal ribosome entry site (ires). Gray boxes, HLF exons; white boxes, 5' and 3' untranslated regions; purple box, puromycin (puro) resistance or truncated EGFR (tEGFR) sequence linked to ZsG by a P2A for optional drug- or antibody-mediated selection. (B-E) Validation of the HLF reporter in human cell lines. HepG2 (HLF expressing) and HEK293 (HLF nonexpressing) cells were electroporated with Cas9/sgRNA RNP either as summarized in panel A or using sgAAVS1 as control. HLF-ZP, rAAV6-encoded HLF repair template driving expression of ZsG and puro resistance. Representative data of 2 independent experiments. (B) Droplet digital polymerase chain reaction (ddPCR) genotyping of targeted cell lines. Black dots represent HR⁻ and red dots represent HR⁺ PCR droplets. HR percentages (red) were calculated as HR⁺ divided by the total number of specific amplicon-containing droplets (black and red). Representative data of 2 independent experiments. (C) ddPCR strategy. External forward primer (ext. FW) binding to a common region outside the 5' HA; 3' reverse (RV) primer amplifying unrecombined locus; ires RV primer amplifying recombined locus; HR⁻ and HR⁺ amplicons are detected by a common FAM-labeled probe, and HR⁺ amplicons are additionally recognized by an HEX-labeled probe that binds to the ires region of the transgene. (D) Fluorescence-activated cell sorting analysis to detect reporter expression. (E) HLF expression levels in selected cell lines. Data curated from Human Protein Atlas.⁵⁰ (F-G) HLF expression in reporter-targeted HepG2 cells. Parental or reporter-targeted/ZsG-sorted HepG2 cells were subjected to quantitative reverse transcription PCR (F) and Western (G) analyses to assess HLF expression. HEK293 cells served as negative control. Asterisks in panel G point to unspecific bands that were used as loading control. HA, homology arm; pA, endogenous HLF polyadenylation signal; TPM, transcripts per kilobase million; WPRE, woodchuck hepatitis virus posttranscriptional response element.