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Deletion of transcription factor binding motifs using the CRISPR/spCas9 system in the β-globin LCR

Transcription factors play roles in gene transcription through direct binding to their motifs in genome, and inhibiting this binding provides an effective strategy for studying their roles. Here, we applied the CRISPR (clustered, regularly interspaced, short palindromic repeat)/spCas9 (CRISPR-associ...

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Autores principales: Kim, Yea Woon, Kim, AeRi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Portland Press Ltd. 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5634328/
https://www.ncbi.nlm.nih.gov/pubmed/28729249
http://dx.doi.org/10.1042/BSR20170976
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author Kim, Yea Woon
Kim, AeRi
author_facet Kim, Yea Woon
Kim, AeRi
author_sort Kim, Yea Woon
collection PubMed
description Transcription factors play roles in gene transcription through direct binding to their motifs in genome, and inhibiting this binding provides an effective strategy for studying their roles. Here, we applied the CRISPR (clustered, regularly interspaced, short palindromic repeat)/spCas9 (CRISPR-associated protein 9) system to mutate the binding motifs of transcription factors. Binding motifs for erythroid-specific transcription factors were mutated in the locus control region (LCR) hypersensitive sites (HSs) of the human β-globin locus. Guide RNAs targetting binding motifs were cloned into lentiviral CRISPR vector containing the spCas9 gene, and transduced into MEL/ch11 cells carrying human chromosome 11. DNA mutations in clonal cells were initially screened by quantitative PCR (qPCR) in genomic DNA and then clarified by sequencing. Mutations in binding motifs reduced occupancy by transcription factors in a chromatin environment. Characterization of mutations revealed that the CRISPR/spCas9 system mainly induced deletions in short regions of <20 bp and preferentially deleted nucleotides around the fifth nucleotide upstream of Protospacer adjacent motifs (PAMs). These results indicate that the CRISPR/Cas9 system is suitable for mutating the binding motifs of transcription factors, and, consequently, would contribute to elucidate the direct roles of transcription factors.
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spelling pubmed-56343282017-10-17 Deletion of transcription factor binding motifs using the CRISPR/spCas9 system in the β-globin LCR Kim, Yea Woon Kim, AeRi Biosci Rep Research Articles Transcription factors play roles in gene transcription through direct binding to their motifs in genome, and inhibiting this binding provides an effective strategy for studying their roles. Here, we applied the CRISPR (clustered, regularly interspaced, short palindromic repeat)/spCas9 (CRISPR-associated protein 9) system to mutate the binding motifs of transcription factors. Binding motifs for erythroid-specific transcription factors were mutated in the locus control region (LCR) hypersensitive sites (HSs) of the human β-globin locus. Guide RNAs targetting binding motifs were cloned into lentiviral CRISPR vector containing the spCas9 gene, and transduced into MEL/ch11 cells carrying human chromosome 11. DNA mutations in clonal cells were initially screened by quantitative PCR (qPCR) in genomic DNA and then clarified by sequencing. Mutations in binding motifs reduced occupancy by transcription factors in a chromatin environment. Characterization of mutations revealed that the CRISPR/spCas9 system mainly induced deletions in short regions of <20 bp and preferentially deleted nucleotides around the fifth nucleotide upstream of Protospacer adjacent motifs (PAMs). These results indicate that the CRISPR/Cas9 system is suitable for mutating the binding motifs of transcription factors, and, consequently, would contribute to elucidate the direct roles of transcription factors. Portland Press Ltd. 2017-08-02 /pmc/articles/PMC5634328/ /pubmed/28729249 http://dx.doi.org/10.1042/BSR20170976 Text en © 2017 The Author(s). http://creativecommons.org/licenses/by/4.0/This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) (http://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Articles
Kim, Yea Woon
Kim, AeRi
Deletion of transcription factor binding motifs using the CRISPR/spCas9 system in the β-globin LCR
title Deletion of transcription factor binding motifs using the CRISPR/spCas9 system in the β-globin LCR
title_full Deletion of transcription factor binding motifs using the CRISPR/spCas9 system in the β-globin LCR
title_fullStr Deletion of transcription factor binding motifs using the CRISPR/spCas9 system in the β-globin LCR
title_full_unstemmed Deletion of transcription factor binding motifs using the CRISPR/spCas9 system in the β-globin LCR
title_short Deletion of transcription factor binding motifs using the CRISPR/spCas9 system in the β-globin LCR
title_sort deletion of transcription factor binding motifs using the crispr/spcas9 system in the β-globin lcr
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5634328/
https://www.ncbi.nlm.nih.gov/pubmed/28729249
http://dx.doi.org/10.1042/BSR20170976
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