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- Title
Tag-seq: a convenient and scalable method for genome-wide specificity assessment of CRISPR/Cas nucleases.
- Authors
Huang, Hongxin; Hu, Yongfei; Huang, Guanjie; Ma, Shufeng; Feng, Jianqi; Wang, Dong; Lin, Ying; Zhou, Jiajian; Rong, Zhili
- Abstract
Genome-wide identification of DNA double-strand breaks (DSBs) induced by CRISPR-associated protein (Cas) systems is vital for profiling the off-target events of Cas nucleases. However, current methods for off-target discovery are tedious and costly, restricting their widespread applications. Here we present an easy alternative method for CRISPR off-target detection by tracing the integrated oligonucleotide Tag using next-generation-sequencing (CRISPR-Tag-seq, or Tag-seq). Tag-seq enables rapid and convenient profiling of nuclease-induced DSBs by incorporating the optimized double-stranded oligodeoxynucleotide sequence (termed Tag), adapters, and PCR primers. Moreover, we employ a one-step procedure for library preparation in Tag-seq, which can be applied in the routine workflow of a molecular biology laboratory. We further show that Tag-seq successfully determines the cleavage specificity of SpCas9 variants and Cas12a/Cpf1 in a large-scale manner, and discover the integration sites of exogenous genes introduced by the Sleeping Beauty transposon. Our results demonstrate that Tag-seq is an efficient and scalable approach to genome-wide identification of Cas-nuclease-induced off-targets. Hongxin Huang and Yongfei Hu et al. develop Tag-seq, a streamlined sequencing method to examine off-target editing by CRISPR-Cas systems. The authors validate Tag-seq in multiple cell lines, demonstrating its broad utility and flexibility in identifying off-target genome editing.
- Subjects
CRISPRS; DOUBLE-strand DNA breaks; GENOME-wide association studies; NUCLEASES; DNA primers; TRANSPOSONS
- Publication
Communications Biology, 2021, Vol 4, Issue 1, p1
- ISSN
2399-3642
- Publication type
Article
- DOI
10.1038/s42003-021-02351-3