Figure 1.

CRISPR-Mediated KI of Hutat2:Fc Gene Fragments into the AAVS1 Locus in Various Cell Lines and Primary Human Monocytes

(A) Schematic of the construction of the donor plasmid and primers design. The homology arms of the donor vector are labeled LR and RR. (B–D) After co-transfection of HeLa cells with sgRNA2 with or without donor plasmids, genomic templates were amplified by PCR using the primers, GT-F/Ai3-2781R (B), Ai3-2737F/Puro-GT-R (C), and Neo-F/GT-R (D), shown in (A). (E) Following selection with Puro, PCR using the primers GT-F/GT-R shown in (A) was performed to semiquantitatively analyze the KI efficiency in HeLa cells transfected with different ratios of sgRNA2 to donor plasmids. The positions of wild-type and transgene chromatids are indicated below and above the gel, respectively. In this assay, HDR resulted in an additional weak PCR band above the wild-type chromatid band. The editing frequencies were calculated and shown below the gel. The percentage of GFP⁺ cells was quantified by FACS, and representative FACS plots are shown directly below the same genotype. (F) As in (E), 293T cells were transfected with the maximum ratio of sgRNA2 and donor plasmids. (G and H) As in (F), U937 cells (G) and primary monocytes (H) were transfected by electroporation but not subjected to selection. KI-293T, CRISPR/Cas9-mediated KI in 293T cells; KI-HeLa, CRISPR/Cas9-mediated KI in HeLa cells; KI-M, CRISPR/Cas9-mediated KI in primary human monocyte; KI-U937, CRISPR/Cas9-mediated KI in U937 cells.