Defining genome-wide CRISPR–Cas genome-editing nuclease activity with GUIDE-seq
Malinin, N.L.; Lee, G.; Lazzarotto, C.R.; Li, Y.; Zheng, Z.; Nguyen, N.T.; Liebers, M.; Topkar, V.V.; Iafrate, A.J.; Le, L.P.; Aryee, M.J.; Joung, J.K.; Tsai, S.Q. (2021). Defining genome-wide CRISPR–Cas genome-editing nuclease activity with GUIDE-seq. Nature Protocols 16(12): 5592-5615. https://dx.doi.org/10.1038/s41596-021-00626-x
In: Nature Protocols. Nature Publishing Group: London. ISSN 1754-2189; e-ISSN 1750-2799, more
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| Authors | | Top |
- Malinin, N.L.
- Lee, G.
- Lazzarotto, C.R.
- Li, Y.
- Zheng, Z.
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- Nguyen, N.T.
- Liebers, M.
- Topkar, V.V.
- Iafrate, A.J.
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- Le, L.P.
- Aryee, M.J.
- Joung, J.K.
- Tsai, S.Q.
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| Abstract |
Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome-editing nucleases in living cells. GUIDE-seq is based on the principle of efficient integration of an end-protected double-stranded oligodeoxynucleotide tag into sites of nuclease-induced DNA double-stranded breaks, followed by amplification of tag-containing genomic DNA molecules and high-throughput sequencing. Here we describe a detailed GUIDE-seq protocol including cell transfection, library preparation, sequencing and bioinformatic analysis. The entire protocol including cell culture can be completed in 9 d. Once tag-integrated genomic DNA is isolated, library preparation, sequencing and analysis can be performed in 3 d. The result is a genome-wide catalog of off-target sites ranked by nuclease activity as measured by GUIDE-seq read counts. GUIDE-seq is one of the most sensitive cell-based methods for defining genome-wide off-target activity and has been broadly adopted for research and therapeutic use. |
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