Figure 2.
CRISPR/Cas9-mediated Cxcr4 inactivation. (A) Sequences and gene locations of 2 sgRNAs that nonspecifically target both the wild-type and WHIM Cxcr4 alleles. sgRNA sequences are in blue and the PAM sequence is in red. (B) Cell-free system. Cxcr4 amplicons generated via PCR were incubated with Cxcr4-sgRNA/Cas9 RNP complex in vitro and the cleavage products were detected by agarose gel electrophoresis. Experimental conditions are indicated at the top of each lane: M, 1 kb DNA ladder markers; no sgRNA, control Cxcr4-amplicon incubated with Cas9 but without sgRNA; Right guide RNA and left guide RNA refer to Cxcr4 amplicons incubated with RNPs containing the indicated sgRNA, each performed in triplicate. ∗ denotes expected fragment sizes based on the sgRNA location within the amplicon. (C-F) Cell-based transfection system using Cxcr4-sgRNA/Cas9 RNP complex. (C-D) Editing of the pre–B-cell lymphoma cell line L1.2. (C) FACS analysis of nonpermeabilized L1.2 cells with Cxcr4 antibody. The sgRNA is specified at the upper left of each panel. Red and orange histograms, Cxcr4-sgRNA/Cas9 RNP-transfected cells tested with isotype control antibody (red) or anti-Cxcr4 antibody (orange). Blue histograms, mock-transfected cells tested with anti-Cxcr4 antibody. (D) T7 endonuclease 1 (T7E1) editing assay of L1.2 cells. Cxcr4 amplicons from mock-transfected cells or cells transfected with either the left or right sgRNA/Cas9 RNP-transfected, as specified at the top of each lane, were treated with T7E1 and the products were revealed via agarose gel electrophoresis. M, 100 bp DNA ladder. Red stars indicate the DNA fragments cleaved by T7E1. (E-G) Editing of primary cKit+ HSPCs from BM of C57BL/6 mice. (E) T7E1 assay. (F) Identification of cleavage site indels. Sequences of 3 Cxcr4 amplicons from edited HSPCs are shown. The PAM sequence is located within the red boxes. Red arrows, deletions of 1 (left) or 2 nucleotides (middle), and insertion of 1 nucleotide (right). (G) Location of premature stop codons in Cxcr4 introduced by CRISPR/Cas9-generated indels. In the DNA sequence, the PAM site is highlighted in red and the sgRNA sequence is in blue. Protein sequence numbers at the top demarcate amino acid positions; red stars show positions of chain termination introduced by the 1 deletion (first star) and 2 deletions or 1 addition (second star).

CRISPR/Cas9-mediated Cxcr4 inactivation. (A) Sequences and gene locations of 2 sgRNAs that nonspecifically target both the wild-type and WHIM Cxcr4 alleles. sgRNA sequences are in blue and the PAM sequence is in red. (B) Cell-free system. Cxcr4 amplicons generated via PCR were incubated with Cxcr4-sgRNA/Cas9 RNP complex in vitro and the cleavage products were detected by agarose gel electrophoresis. Experimental conditions are indicated at the top of each lane: M, 1 kb DNA ladder markers; no sgRNA, control Cxcr4-amplicon incubated with Cas9 but without sgRNA; Right guide RNA and left guide RNA refer to Cxcr4 amplicons incubated with RNPs containing the indicated sgRNA, each performed in triplicate. ∗ denotes expected fragment sizes based on the sgRNA location within the amplicon. (C-F) Cell-based transfection system using Cxcr4-sgRNA/Cas9 RNP complex. (C-D) Editing of the pre–B-cell lymphoma cell line L1.2. (C) FACS analysis of nonpermeabilized L1.2 cells with Cxcr4 antibody. The sgRNA is specified at the upper left of each panel. Red and orange histograms, Cxcr4-sgRNA/Cas9 RNP-transfected cells tested with isotype control antibody (red) or anti-Cxcr4 antibody (orange). Blue histograms, mock-transfected cells tested with anti-Cxcr4 antibody. (D) T7 endonuclease 1 (T7E1) editing assay of L1.2 cells. Cxcr4 amplicons from mock-transfected cells or cells transfected with either the left or right sgRNA/Cas9 RNP-transfected, as specified at the top of each lane, were treated with T7E1 and the products were revealed via agarose gel electrophoresis. M, 100 bp DNA ladder. Red stars indicate the DNA fragments cleaved by T7E1. (E-G) Editing of primary cKit+ HSPCs from BM of C57BL/6 mice. (E) T7E1 assay. (F) Identification of cleavage site indels. Sequences of 3 Cxcr4 amplicons from edited HSPCs are shown. The PAM sequence is located within the red boxes. Red arrows, deletions of 1 (left) or 2 nucleotides (middle), and insertion of 1 nucleotide (right). (G) Location of premature stop codons in Cxcr4 introduced by CRISPR/Cas9-generated indels. In the DNA sequence, the PAM site is highlighted in red and the sgRNA sequence is in blue. Protein sequence numbers at the top demarcate amino acid positions; red stars show positions of chain termination introduced by the 1 deletion (first star) and 2 deletions or 1 addition (second star).

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