Defining genome-wide CRISPR-Cas genome-editing nuclease activity with GUIDE-seq.
CRISPR-Associated Protein 9
/ genetics
CRISPR-Cas Systems
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats
DNA Primers
/ chemical synthesis
Deoxyribonucleases, Type II Site-Specific
/ chemistry
Electroporation
/ methods
Gene Editing
/ methods
Genome, Human
Humans
Osteoblasts
/ cytology
Plasmids
/ chemistry
Polymerase Chain Reaction
/ methods
Primary Cell Culture
RNA, Guide, Kinetoplastida
/ genetics
T-Lymphocytes
/ cytology
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
12 2021
12 2021
Historique:
received:
31
10
2020
accepted:
02
09
2021
pubmed:
14
11
2021
medline:
24
12
2021
entrez:
13
11
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 34773119
doi: 10.1038/s41596-021-00626-x
pii: 10.1038/s41596-021-00626-x
pmc: PMC9331158
mid: NIHMS1811119
doi:
Substances chimiques
DNA Primers
0
RNA, Guide
0
CRISPR-Associated Protein 9
EC 3.1.-
Cas9 endonuclease Streptococcus pyogenes
EC 3.1.-
endodeoxyribonuclease NdeI
EC 3.1.21.-
Deoxyribonucleases, Type II Site-Specific
EC 3.1.21.4
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Review
Langues
eng
Sous-ensembles de citation
IM
Pagination
5592-5615Subventions
Organisme : NIGMS NIH HHS
ID : DP1 GM105378
Pays : United States
Organisme : Doris Duke Charitable Foundation
ID : 2020154
Pays : United States
Organisme : NIAID NIH HHS
ID : U01 AI157189
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM088040
Pays : United States
Organisme : NIAMS NIH HHS
ID : R01 AR063070
Pays : United States
Organisme : Doris Duke Charitable Foundation
ID : 2017093
Pays : United States
Informations de copyright
© 2021. The Author(s), under exclusive licence to Springer Nature Limited.
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