Fig 1.
Evaluation of CRISPR/Cas9 mediated cleavage of VDRT1 and VDRT2 in human 293T cells.
(A) Schematic diagram of location of two VDR targeting sites in human chromosome. The red lines stand for the locations of two target sites in VDR. The primers for the PCR amplified target sites sequence are indicated by a black arrow. (B) The efficiency of CRISPR/Cas9 mediated cleavage at two target sites in HEK293T cells. (C) CRISPR/Cas9 mediated VDR targeted editing in human 293T cells. Target sequences were highlighted with pink boxes, and PAM sequences were highlighted as underlined. Green boxes stand for insertion sequences (▼, deletion junction; △, deletion; +, insertion). (D) The large fragment deletion was identified by PCR. Left panel, the PCR products of lane 2 (+CRISPR/Cas9) and lane 4 (WT-DNA) were amplified from treated cells and wild type genomic DNA, respectively. Right panel, digital PCR analysis of the large genomic fragment deletions within the VDR. The strong 400bp band in all lanes W1-W11 (on Mark right) is a non-specific amplification product. The 500bp bands from D1-D11 (on Mark right) correspond to the expected size of the PCR product in the event of large fragment deletion of the intervening sequence. (E) Target sequences were highlighted with red boxes and pink boxes, and PAM sequences were highlighted as underlined. Green boxes stand for insertion sequences (▼, deletion junction).
Table 1.
Summary of production of VDR gene modified mice via CRISPR/Cas9.
Fig 2.
Detection of CRISPR/Cas9 mediated modifications of VDR in mice.
(A) PCR products of the targeted region of VDRT1 from founder mice. (B) Detection of cleavage of VDRT1 by T7E1 assay. (C) Sequencing results of re-edited VDRT1 loci detected in founder mice.
Fig 3.
Expression levels of VDR, Cyp27B1 and Cyp24A1 in different tissues from founder mice.
(A) Phenotypes of VDR knock-out mice with different ages. (i,ii,iii) VDR-/- mouse #4 at two, three and four months old, respectively; (iv) VDR-/- mice #26,#9 and VDR-/+ #17 mouse at four months old; (v) wild type mice at four months old. (B) immunofluorescence histochemistry of VDR in mouse small intestine and muscle. Up panel, D-6/DAPI colocalization in the sections of small intestine. Down panel, D-6/DAPI colocalization in the sections of muscle. (C) The differential expression of Cyp27B1 of kidney, skin, small intestine and muscle by qPCR between VDR knockout mice and WT mice. (D) The differential expression of Cyp24A1 in mouse kidney, skin, small intestine and muscle.
Fig 4.
Detection of the CRISPR/Cas9 mediated off-target cleavages in vivo.
(A) PCR products of the potential off-target sites from founder mice. OT1 and OT5 were selected and amplified from genomic DNA of VDR knockout mice. (B) Detection of off-target cleavage by VDRT1 sgRNA at OT1 and OT5 by T7E1 assay. (C) Sequencing results of PCR products.