Peking University Yi Chengqi research group develops a new tool for off-target detection of cytosine base editor

On June 8, 2021, the Peking University-Tsinghua Life Sciences Joint Center and the Peking University School of Life Sciences Professor Yi Chengqi’s research group published in Nature Methods the title " Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors " cover article

Established an unbiased off-target detection technology

The author captured the intermediate (dU) produced by the cytosine base editor CBE[1], and performed a series of labeling and enrichment [2, 3] to obtain off-target signals with continuous C-to-T mutations.

Figure 1 Detect-seq technology roadmap

Using Detect-seq technology to discover a large number of off-target sites on the whole genome caused by CBE tools

The researchers transfected human embryonic kidney cells HEK293T and human breast cancer cells MCF-7 into BE4max[4], and used a variety of different sgRNAs to perform C-to-T editing on the targeted sites of the genome; at the same time, these samples were performed Detect-seq detection

Figure 2 Cas-dependent off-target sites identified in three different sgRNA samples, where the blue circle represents off-targets, and the red square represents on-target EMX1n=48.

Two new off-target sites were discovered (out-of-protospacer edit and target-strand edit)

Interestingly, the researchers also discovered two new Cas-dependent off-target edits based on the Detect-seq technology: out-of-protospacer edit and target-strand edit.

Figure 3 Mode diagram of editing outside the binding region of sgRNA and targeting strand editing

In summary, the Detect-seq technology provides a highly sensitive, highly specific, and non-biased method for detecting CBE off-target sites in cells in the field of base editing

Yi Chengqi is the corresponding author of this article; Dr.

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