Massively targeted evaluation of therapeutic CRISPR off-targets in cells

Xiaoguang Pan, Kunli Qu, Hao Yuan, Xi Xiang, Christian Anthon, Liubov Pashkova, Xue Liang, Peng Han, Giulia I. Corsi, Fengping Xu, Ping Liu, Jiayan Zhong, Yan Zhou, Tao Ma, Hui Jiang, Junnian Liu, Jian Wang, Niels Jessen, Lars Bolund, Huanming Yang, Xun Xu, George M. Church (), Jan Gorodkin (), Lin Lin () and Yonglun Luo ()
Additional contact information
Xiaoguang Pan: Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
Kunli Qu: Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
Hao Yuan: Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
Xi Xiang: Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
Christian Anthon: University of Copenhagen
Liubov Pashkova: University of Copenhagen
Xue Liang: Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
Peng Han: Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
Giulia I. Corsi: University of Copenhagen
Fengping Xu: Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
Ping Liu: BGI-Research, BGI-Shenzhen
Jiayan Zhong: BGI-Research, BGI-Shenzhen
Yan Zhou: Aarhus University
Tao Ma: BGI-Research, BGI-Shenzhen
Hui Jiang: BGI-Research, BGI-Shenzhen
Junnian Liu: Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
Jian Wang: BGI-Research, BGI-Shenzhen
Niels Jessen: Aarhus University
Lars Bolund: Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
Huanming Yang: BGI-Research, BGI-Shenzhen
Xun Xu: BGI-Research, BGI-Shenzhen
George M. Church: Harvard Medical School
Jan Gorodkin: University of Copenhagen
Lin Lin: Aarhus University
Yonglun Luo: Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen

Nature Communications, 2022, vol. 13, issue 1, 1-14

Abstract: Abstract Methods for sensitive and high-throughput evaluation of CRISPR RNA-guided nucleases (RGNs) off-targets (OTs) are essential for advancing RGN-based gene therapies. Here we report SURRO-seq for simultaneously evaluating thousands of therapeutic RGN OTs in cells. SURRO-seq captures RGN-induced indels in cells by pooled lentiviral OTs libraries and deep sequencing, an approach comparable and complementary to OTs detection by T7 endonuclease 1, GUIDE-seq, and CIRCLE-seq. Application of SURRO-seq to 8150 OTs from 110 therapeutic RGNs identifies significantly detectable indels in 783 OTs, of which 37 OTs are found in cancer genes and 23 OTs are further validated in five human cell lines by targeted amplicon sequencing. Finally, SURRO-seq reveals that thermodynamically stable wobble base pair (rG•dT) and free binding energy strongly affect RGN specificity. Our study emphasizes the necessity of thoroughly evaluating therapeutic RGN OTs to minimize inevitable off-target effects.

Date: 2022
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DOI: 10.1038/s41467-022-31543-6

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