Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen, Qingdao, China.
Department of Biology, Copenhagen University, Copenhagen, Denmark.
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China.
Center for non-coding RNA in Technology and Health, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark.
BGI-Research, BGI-Shenzhen, Shenzhen, China.
MGI, BGI-Shenzhen, Shenzhen, China.
Steno Diabetes Center Aarhus, Aarhus University Hospital, Aarhus, Denmark.
IBMC-BGI Center, the Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou, Zhejiang, 310022, China.
Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, Shenzhen, China.
Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA. gchurch@genetics.med.harvard.edu.
Center for non-coding RNA in Technology and Health, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark. gorodkin@rth.dk.
Department of Biomedicine, Aarhus University, Aarhus, Denmark. lin.lin@biomed.au.dk.
Steno Diabetes Center Aarhus, Aarhus University Hospital, Aarhus, Denmark. lin.lin@biomed.au.dk.
Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen, Qingdao, China. alun@biomed.au.dk.
Department of Biomedicine, Aarhus University, Aarhus, Denmark. alun@biomed.au.dk.
College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China. alun@biomed.au.dk.
Steno Diabetes Center Aarhus, Aarhus University Hospital, Aarhus, Denmark. alun@biomed.au.dk.
IBMC-BGI Center, the Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou, Zhejiang, 310022, China. alun@biomed.au.dk.
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.
