Figure 5

Gene knockin using acoustic-transfection and CRISPR-Cas9. (A) Schematic diagram of the targeting for the PPP1R12C gene in the AAVS1 locus. The three solid circles indicate first 3 exons of PPP1R12C. The dark grey boxes with Puro, LifeAct, and EGFP indicate the genomic PCR regions, thereby confirming the correct HDR events by CRISPR-Cas9, where the results are shown in (B). A solid arrow head represents the genomic cut by the CRISPR-Cas9. The 20-bp gRNA target and PAM sequences are also shown. Two types of donor repair templates for CRISPR-Cas9 targeting the locus are shown. SA-2A and PURO is the splice acceptor sequence followed by a 2A self-cleaving peptide sequence and the puromycin resistance gene. The pA is the polyadenylation sequence. The pCAG-LifeAct-TagRFP-SV40pA sequence is used to target the F-actin with a fluorescence marker (TagRFP) and pCAGGS-EGFP-pA is used to target the cytoplasm with EGFP. We cloned AAV-LifeAct-TagRFP and used AAV-CAGGS-EGFP from Addgene (#22212) as a control. To develop the AAV-LifeAct-TagRFP donor repair template, the pCAGGS-EGFP-pA sequence from AAV-CAGGS-EGFP was replaced with the pCAG-LifeAct-TagRFP-SV40pA sequence. The same gRNA and hCas9 were used for both CRISPR-Cas9 systems. (B) Arrows on the left indicate the expected positions of the DNA bands by genomic PCR. (C) Acoustic-transfection and (D) lipofectamine 3000 (as a control) were used to deliver two types of CRISPR-Cas9 systems. Representative images of the HeLa cells with gene expressions after the HDR by AAV-LifeAct-TagRFP (first panel) and AAV-CAGGS-EGFP (second panel) donor templates are presented in (C) and (D). Scale bars indicate 20 μm.