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| Status |
Public on Jun 17, 2022 |
| Title |
Massively targeted evaluation of therapeutic CRISPR off-targets in cells |
| Organism |
Homo sapiens |
| Experiment type |
Other
|
| Summary |
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.
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| |
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| Overall design |
SURRO-seq analysis on 12K off-target library
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| |
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| Contributor(s) |
Pan X, Qu K, Lin L, Luo Y |
| Citation(s) |
35831290 |
| |
| Submission date |
Jun 17, 2022 |
| Last update date |
Jul 20, 2022 |
| Contact name |
Lin Lin |
| E-mail(s) |
lin.lin@biomed.au.dk
|
| Organization name |
Aarhus University
|
| Department |
Department of Biomedicine
|
| Street address |
Høegh-Guldbergs Gade 10
|
| City |
Aarhus |
| ZIP/Postal code |
8000 |
| Country |
Denmark |
| |
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| Platforms (1) |
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| Samples (702)
|
|
| Relations |
| BioProject |
PRJNA850372 |
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