Fig. 4

Isoform-specific dual-sgRNA CRISPR-mediated CXCL12γ KO. a Schematic representation of the CRISPR-induced deletion of the fourth exon (C-terminal tail) of CXCL12γ. sgRNA#1 was designed to target upstream of the fourth exon of CXCL12γ, and sgRNA#2 was designed to target the 3′UTR of the fourth exon. Rectangles represent exons, and filled rectangles represent coding sequences. Lines indicate introns. (Right) PCR analysis of the deletion of CXCL12γ; primers used are as indicated in panel A. Genomic DNA was isolated from HS5 cells with the CRISPR empty vector (WT) or co-transduced with CRISPR sgRNA#1 and CRISPR sgRNA#2, either before single-cell cloning (pool) or from two single-cell KO clones (CXCL12γKO#1 and #2). Water was used as negative control. DNA ladder size is indicated on the left. b Confirmation of CRISPR-induced deletion by Sanger sequencing. Genomic DNA was isolated from HS5 cells transduced with empty vector CRISPR (HS5-WT) or CRISPR sgRNA#1 and CRISPR sgRNA#2 (HS5-CXCL12γKO). The CRISPR cutting sites are indicated by vertical line in HS5-WT; c Confirmation of CXCL12γ deletion by flow cytometry. Cell surface expression of CXCL12γ on HS5 cells transduced with either empty vector CRISPR (HS5-WT) or HS5-CXCL12γKO which were stained with the CXCL12γ-specific mAB 6E9