No off-target mutations are detected by a sensitive assay in CD34⁺HSPCs after editing at the γ-globin gene promoters. Purified genomic DNA from normal CD34⁺ cell–derived erythroblasts was analyzed by the CIRCLE-seq method for defining Cas9 genome-wide activity in vitro. The left panel shows the 25 most frequent candidate off-target sites for Cas9:sgRNA-1 RNP identified by CIRCLE-seq analysis on purified genomic DNA. The on-target sites including sgRNA and PAM sequences are shown at the top. Off-target sites are ordered by CIRCLE-seq read count, with matches to the intended target site shown as dots and mismatches shown as colored nucleotides. The right panel shows the indel frequencies at candidate off-target sites validated by targeted high-throughput sequencing of CD34⁺ HSPCs edited with Cas9:sgRNA-1 RNP using the Neon Transfection System (blue squares) and control nonedited CD34⁺ cells (red circles). The threshold of detection for NGS is ∼0.01% to 0.1%.