Figure 3. CRISPR-Gold induces HDR in vitro.

a) CRISPR-Gold can induce HDR in BFP-HEK cells by delivering Cas9 RNP and a donor DNA. 11.3% of BFP-HEK cells were converted to GFP expressing cells, after treatment with CRISPR-Gold. GFP expression due to BFP sequence editing was determined by flow cytometry.

b) The uptake of CRISPR-Gold in HEK293 cells is dependent on non-clathrin mediated endocytosis. CRISPR-Gold containing Alexa647 labeled sgRNA was added to HEK293 cells and the uptake efficiency was measured using flow cytometry. Various inhibitors and low temperature were used to study the cellular uptake mechanism. Also, the uptake of CRISPR-Gold without the PAsp(DET) polymer coating was investigated. Genistein, MBCD, and 4°C all significantly inhibited the uptake of CRISPR-Gold, whereas clathrin based endocytosis inhibitors had no effect. Mean ± S.E, n=3. **, p < 0.01.

c) Non-clathrin mediated endocytosis plays a critical role in CRISPR-Gold mediated HDR. The inhibitor of clathrin mediated endocytosis, chlorpromazine, has minimal effects on CRISPR-Gold mediated HDR, whereas the caveolae/raft mediated endocytosis inhibitor MBCD and genistein reduced CRISPR-Gold mediated HDR by 80%. Mean ± S.E, n=3. **, p < 0.01, ns= not significant.

d) CRISPR-Gold induces HDR in hES and hiPS cells with higher efficiency than lipofectamine or nucleofection. Cells were treated with CRISPR-Gold that contained a donor sequence with a HindIII cleavage site, and a guide RNA that targeted the CXCR4 gene. The CXCR4 gene was amplified by PCR and the HDR efficiency was determined by quantifying HindIII cleavage of the CXCR4 PCR amplicon. Mean ± S.E, n=3. A one-way ANOVA test had a p= 0.0066 for hES samples and p=0.0023 for hiPS samples.

e) Sanger sequencing demonstrates that CRISPR-Gold induces HDR in hES cells. PCR of the CXCR4 gene was performed on CRISPR-Gold treated hES cells, and sequencing confirms the presence of the 12 bp insertion (pink box).