Defining genome-wide CRISPR-Cas genome-editing nuclease activity with GUIDE-seq.

Autor: Malinin NL; Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA., Lee G; Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA., Lazzarotto CR; Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA., Li Y; Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA., Zheng Z; Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China., Nguyen NT; Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA.; Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital, and Harvard Medical School, Boston, MA, USA., Liebers M; Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA., Topkar VV; Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA.; Biophysics Program, Stanford University, Stanford, CA, USA., Iafrate AJ; Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA., Le LP; Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA., Aryee MJ; Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA., Joung JK; Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA., Tsai SQ; Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA. shengdar.tsai@stjude.org.
Jazyk: angličtina
Zdroj: Nature protocols [Nat Protoc] 2021 Dec; Vol. 16 (12), pp. 5592-5615. Date of Electronic Publication: 2021 Nov 12.
DOI: 10.1038/s41596-021-00626-x
Abstrakt: 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.
(© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)
Databáze: MEDLINE
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