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Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9

BACKGROUND: Krüppel Associated Box-containing Zinc Finger Proteins (KRAB-ZFPs), representing the largest superfamily of transcription factors in mammals, are predicted to primarily target and repress transposable elements (TEs). It is challenging to dissect the distinct functions of these transcript...

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Autores principales: Zhang, Yang, He, Fei, Zhang, Yanning, Dai, Qian, Li, Qintong, Nan, Jing, Miao, Ruidong, Cheng, Bo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9647903/
https://www.ncbi.nlm.nih.gov/pubmed/36357895
http://dx.doi.org/10.1186/s13100-022-00279-x
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author Zhang, Yang
He, Fei
Zhang, Yanning
Dai, Qian
Li, Qintong
Nan, Jing
Miao, Ruidong
Cheng, Bo
author_facet Zhang, Yang
He, Fei
Zhang, Yanning
Dai, Qian
Li, Qintong
Nan, Jing
Miao, Ruidong
Cheng, Bo
author_sort Zhang, Yang
collection PubMed
description BACKGROUND: Krüppel Associated Box-containing Zinc Finger Proteins (KRAB-ZFPs), representing the largest superfamily of transcription factors in mammals, are predicted to primarily target and repress transposable elements (TEs). It is challenging to dissect the distinct functions of these transcription regulators due to their sequence similarity and diversity, and also the complicated repetitiveness of their targeting TE sequences. RESULTS: Mouse KRAB-Zfps are mainly organized into clusters genomewide. In this study, we revealed that the intra-cluster members had a close evolutionary relationship, and a similar preference for zinc finger (ZnF) usage. KRAB-Zfps were expressed in a cell type- or tissue type specific manner and they tended to be actively transcribed together with other cluster members. Further sequence analyses pointed out the linker sequences in between ZnFs were conserved, and meanwhile had distinct cluster specificity. Based on these unique characteristics of KRAB-Zfp clusters, sgRNAs were designed to edit cluster-specific linkers to abolish the functions of the targeted cluster(s). Using mouse embryonic stem cells (mESC) as a model, we screened and obtained a series of sgRNAs targeting various highly expressed KRAB-Zfp clusters. The effectiveness of sgRNAs were verified in a reporter assay exclusively developed for multi-target sgRNAs and further confirmed by PCR-based analyses. Using mESC cell lines inducibly expressing Cas9 and these sgRNAs, we found that editing different KRAB-Zfp clusters resulted in the transcriptional changes of distinct categories of TEs. CONCLUSIONS: Collectively, the intrinsic sequence correlations of intra-cluster KRAB-Zfp members discovered in this study suggest that the conserved cluster specific linkers played crucial roles in diversifying the tandem ZnF array and the related target specificity of KRAB-Zfps during clusters’ evolution. On this basis, an effective CRISPR-Cas9 based approach against the linker sequences is developed and verified for rapidly editing KRAB-Zfp clusters to identify the regulatory correlation between the cluster members and their potential TE targets. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13100-022-00279-x.
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spelling pubmed-96479032022-11-15 Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9 Zhang, Yang He, Fei Zhang, Yanning Dai, Qian Li, Qintong Nan, Jing Miao, Ruidong Cheng, Bo Mob DNA Research BACKGROUND: Krüppel Associated Box-containing Zinc Finger Proteins (KRAB-ZFPs), representing the largest superfamily of transcription factors in mammals, are predicted to primarily target and repress transposable elements (TEs). It is challenging to dissect the distinct functions of these transcription regulators due to their sequence similarity and diversity, and also the complicated repetitiveness of their targeting TE sequences. RESULTS: Mouse KRAB-Zfps are mainly organized into clusters genomewide. In this study, we revealed that the intra-cluster members had a close evolutionary relationship, and a similar preference for zinc finger (ZnF) usage. KRAB-Zfps were expressed in a cell type- or tissue type specific manner and they tended to be actively transcribed together with other cluster members. Further sequence analyses pointed out the linker sequences in between ZnFs were conserved, and meanwhile had distinct cluster specificity. Based on these unique characteristics of KRAB-Zfp clusters, sgRNAs were designed to edit cluster-specific linkers to abolish the functions of the targeted cluster(s). Using mouse embryonic stem cells (mESC) as a model, we screened and obtained a series of sgRNAs targeting various highly expressed KRAB-Zfp clusters. The effectiveness of sgRNAs were verified in a reporter assay exclusively developed for multi-target sgRNAs and further confirmed by PCR-based analyses. Using mESC cell lines inducibly expressing Cas9 and these sgRNAs, we found that editing different KRAB-Zfp clusters resulted in the transcriptional changes of distinct categories of TEs. CONCLUSIONS: Collectively, the intrinsic sequence correlations of intra-cluster KRAB-Zfp members discovered in this study suggest that the conserved cluster specific linkers played crucial roles in diversifying the tandem ZnF array and the related target specificity of KRAB-Zfps during clusters’ evolution. On this basis, an effective CRISPR-Cas9 based approach against the linker sequences is developed and verified for rapidly editing KRAB-Zfp clusters to identify the regulatory correlation between the cluster members and their potential TE targets. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13100-022-00279-x. BioMed Central 2022-11-10 /pmc/articles/PMC9647903/ /pubmed/36357895 http://dx.doi.org/10.1186/s13100-022-00279-x Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Zhang, Yang
He, Fei
Zhang, Yanning
Dai, Qian
Li, Qintong
Nan, Jing
Miao, Ruidong
Cheng, Bo
Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9
title Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9
title_full Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9
title_fullStr Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9
title_full_unstemmed Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9
title_short Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9
title_sort exploration of the regulatory relationship between krab-zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by crispr-cas9
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9647903/
https://www.ncbi.nlm.nih.gov/pubmed/36357895
http://dx.doi.org/10.1186/s13100-022-00279-x
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