Massively targeted evaluation of therapeutic CRISPR off-targets in cells
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Massively targeted evaluation of therapeutic CRISPR off-targets in cells. / Pan, Xiaoguang; Qu, Kunli; Yuan, Hao; Xiang, Xi; Anthon, Christian; Pashkova, Liubov; Liang, Xue; Han, Peng; Corsi, Giulia I.; Xu, Fengping; Liu, Ping; Zhong, Jiayan; Zhou, Yan; Ma, Tao; Jiang, Hui; Liu, Junnian; Wang, Jian; Jessen, Niels; Bolund, Lars; Yang, Huanming; Xu, Xun; Church, George M.; Gorodkin, Jan; Lin, Lin; Luo, Yonglun.
In: Nature Communications, Vol. 13, 4049, 2022.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Massively targeted evaluation of therapeutic CRISPR off-targets in cells
AU - Pan, Xiaoguang
AU - Qu, Kunli
AU - Yuan, Hao
AU - Xiang, Xi
AU - Anthon, Christian
AU - Pashkova, Liubov
AU - Liang, Xue
AU - Han, Peng
AU - Corsi, Giulia I.
AU - Xu, Fengping
AU - Liu, Ping
AU - Zhong, Jiayan
AU - Zhou, Yan
AU - Ma, Tao
AU - Jiang, Hui
AU - Liu, Junnian
AU - Wang, Jian
AU - Jessen, Niels
AU - Bolund, Lars
AU - Yang, Huanming
AU - Xu, Xun
AU - Church, George M.
AU - Gorodkin, Jan
AU - Lin, Lin
AU - Luo, Yonglun
N1 - Publisher Copyright: © 2022, The Author(s).
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
U2 - 10.1038/s41467-022-31543-6
DO - 10.1038/s41467-022-31543-6
M3 - Journal article
C2 - 35831290
AN - SCOPUS:85133983151
VL - 13
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
M1 - 4049
ER -
ID: 315990226