Detection of intergenic deletion. (A) The detection of the intergenic 4.9-k deletion was performed as described previously.^27,38  Genomic DNA isolated from total bone marrow cells was used as a template. A 9.9-kb genomic region spanning the 2 sgHBG-2 mapping sites within the HBG1 and HBG2 promoters was amplified by PCR. An additional 5.0-kb band in the product indicates the occurrence of the 4.9-k deletion. The percentage of deletion was calculated according to a standard curve formula that was generated by PCR of templates with defined ratios of the 4.9-k deletion. Samples derived from mice in vivo transduced with an HDAd-HBG-CRISPR vector²⁷  targeting the HBG1/2 promoter were used for comparison. Each lane represents 1 animal. (B) Summary of the percentage of deletion of 4.9 k in panel A. Each symbol represents 1 animal. *P < .05. (C) Measurement of off-target editing by T7EI cleavage assay. The 10 top-scoring, off-target sites in the whole-mouse genome (mOTS-1 to -10; supplemental Tables 1 and 3) were amplified from genomic DNA of bone marrow cells. Samples from nontransduced mice were used as the negative control. The amplicon size and predicted bands after cleavage are listed below the gel lanes. On-target cleavage at the HBG1/2 promoter is also included. The percentage of cleavage is also shown below the lanes. ND, not detected. DNA ladder sizes are indicated on the left of the figure.