A reverse chromatin immunoprecipitation technique based on the CRISPR-dCas9 system.
Journal
Plant physiology
ISSN: 1532-2548
Titre abrégé: Plant Physiol
Pays: United States
ID NLM: 0401224
Informations de publication
Date de publication:
17 03 2023
17 03 2023
Historique:
received:
20
09
2022
accepted:
12
10
2022
pubmed:
29
10
2022
medline:
22
3
2023
entrez:
28
10
2022
Statut:
ppublish
Résumé
DNA-protein interaction is one of the most crucial interactions in biological processes. However, the technologies available to study DNA-protein interactions are all based on DNA hybridization; however, DNA hybridization is not highly specific and is relatively low in efficiency. RNA-guided DNA recognition is highly specific and efficient. To overcome the limitations of technologies based on DNA hybridization, we built a DNA-binding protein capture technology based on the clustered regularly interspaced palindromic repeats (CRISPR)-dead Cas9 (dCas9) system and transient genetic transformation, termed reverse chromatin immunoprecipitation based on CRISPR-dCas9 system (R-ChIP-dCas9). In this system, dCas9 was fused with Strep-Tag II to form a fusion protein for StrepTactin affinity purification. Transient transformation was performed for the expression of dCas9 and guide RNA (gRNA) to form the dCas9-gRNA complex in birch (Betula platyphylla) plants, which binds to the target genomic DNA region. The dCas9-gRNA-DNA complex was crosslinked, then the chromatin was sonicated into fragments, and purified using StrepTactin beads. The proteins binding to the target genomic DNA region were identified using mass spectrometry. Using this method, we determined the upstream regulators of a NAM, ATAF, and CUC (NAC) transcription factor (TF), BpNAC090, and 32 TFs potentially regulating BpNAC090 were identified. The reliability of R-ChIP-dCas9 was further confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assays, and yeast one-hybrid. This technology can be adapted to various plant species and does not depend on the availability of a stable transformation system; therefore, it has wide application in identifying proteins bound to genomic DNA.
Identifiants
pubmed: 36305686
pii: 6777272
doi: 10.1093/plphys/kiac506
pmc: PMC10022611
doi:
Substances chimiques
DNA
9007-49-2
RNA
63231-63-0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1505-1519Commentaires et corrections
Type : CommentIn
Informations de copyright
© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Déclaration de conflit d'intérêts
Conflict of interest statement. All authors declared there are no confilcts of interest.
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