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; Yonglun Luo

doi:10.1038/s41467-022-31543-6

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, Yonglun Luo

Research output: Contribution to journal › Article › peer-review

30 Scopus citations

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.

Original language	English
Article number	4049
Journal	Nature Communications
Volume	13
Issue number	1
DOIs	https://doi.org/10.1038/s41467-022-31543-6
State	Published - Dec 2022
Externally published	Yes

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10.1038/s41467-022-31543-6

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Pan, X., Qu, K., Yuan, H., Xiang, X., Anthon, C., Pashkova, L., Liang, X., Han, P., Corsi, G. I., Xu, F., Liu, P., Zhong, J., Zhou, Y., Ma, T., Jiang, H., Liu, J., Wang, J., Jessen, N., Bolund, L., ... Luo, Y. (2022). Massively targeted evaluation of therapeutic CRISPR off-targets in cells. Nature Communications, 13(1), Article 4049. https://doi.org/10.1038/s41467-022-31543-6

@article{748cf73287d544be8ec6fb2594b6b39d,

title = "Massively targeted evaluation of therapeutic CRISPR off-targets in cells",

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.",

author = "Xiaoguang Pan and Kunli Qu and Hao Yuan and Xi Xiang and Christian Anthon and Liubov Pashkova and Xue Liang and Peng Han and Corsi, \{Giulia I.\} and Fengping Xu and Ping Liu and Jiayan Zhong and Yan Zhou and Tao Ma and Hui Jiang and Junnian Liu and Jian Wang and Niels Jessen and Lars Bolund and Huanming Yang and Xun Xu and Church, \{George M.\} and Jan Gorodkin and Lin Lin and Yonglun Luo",

note = "Publisher Copyright: {\textcopyright} 2022, The Author(s).",

year = "2022",

month = dec,

doi = "10.1038/s41467-022-31543-6",

language = "英语",

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Pan, X, Qu, K, Yuan, H, Xiang, X, Anthon, C, Pashkova, L, Liang, X, Han, P, Corsi, GI, Xu, F, Liu, P, Zhong, J, Zhou, Y, Ma, T, Jiang, H, Liu, J, Wang, J, Jessen, N, Bolund, L, Yang, H, Xu, X, Church, GM, Gorodkin, J, Lin, L & Luo, Y 2022, 'Massively targeted evaluation of therapeutic CRISPR off-targets in cells', Nature Communications, vol. 13, no. 1, 4049. https://doi.org/10.1038/s41467-022-31543-6

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

PY - 2022/12

Y1 - 2022/12

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.

UR - https://www.scopus.com/pages/publications/85133983151

U2 - 10.1038/s41467-022-31543-6

DO - 10.1038/s41467-022-31543-6

M3 - 文章

C2 - 35831290

AN - SCOPUS:85133983151

SN - 2041-1723

VL - 13

JO - Nature Communications

JF - Nature Communications

IS - 1

M1 - 4049

ER -

Massively targeted evaluation of therapeutic CRISPR off-targets in cells

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