Cargando…

Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9

The human genome contains millions of DNA regulatory elements and a large number of gene clusters, most of which have not been tested experimentally. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) programed with a synthetic single-guide RNA...

Descripción completa

Detalles Bibliográficos
Autores principales: Li, Jinhuan, Shou, Jia, Guo, Ya, Tang, Yuanxiao, Wu, Yonghu, Jia, Zhilian, Zhai, Yanan, Chen, Zhifeng, Xu, Quan, Wu, Qiang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524425/
https://www.ncbi.nlm.nih.gov/pubmed/25757625
http://dx.doi.org/10.1093/jmcb/mjv016
_version_ 1782384192879853568
author Li, Jinhuan
Shou, Jia
Guo, Ya
Tang, Yuanxiao
Wu, Yonghu
Jia, Zhilian
Zhai, Yanan
Chen, Zhifeng
Xu, Quan
Wu, Qiang
author_facet Li, Jinhuan
Shou, Jia
Guo, Ya
Tang, Yuanxiao
Wu, Yonghu
Jia, Zhilian
Zhai, Yanan
Chen, Zhifeng
Xu, Quan
Wu, Qiang
author_sort Li, Jinhuan
collection PubMed
description The human genome contains millions of DNA regulatory elements and a large number of gene clusters, most of which have not been tested experimentally. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) programed with a synthetic single-guide RNA (sgRNA) emerges as a method for genome editing in virtually any organisms. Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs. Specifically, we found that, in cultured human cells and mice, efficient precise inversions of DNA fragments ranging in size from a few tens of bp to hundreds of kb could be generated. In addition, DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks (DSBs) on two homologous chromosomes (chromatids). Moreover, junctions of combinatorial inversions and duplications of the protocadherin (Pcdh) gene clusters induced by Cas9 with four sgRNAs could be detected. In mice, we obtained founders with alleles of precise inversions, duplications, and deletions of DNA fragments of variable sizes by CRISPR. Interestingly, we found that very efficient inversions were mediated by microhomology-mediated end joining (MMEJ) through short inverted repeats. We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice. Finally, we applied this CRISPR method to a regulatory element of the Pcdhα cluster and found a new role in the regulation of members of the Pcdhγ cluster. This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters.
format Online
Article
Text
id pubmed-4524425
institution National Center for Biotechnology Information
language English
publishDate 2015
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-45244252015-08-07 Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9 Li, Jinhuan Shou, Jia Guo, Ya Tang, Yuanxiao Wu, Yonghu Jia, Zhilian Zhai, Yanan Chen, Zhifeng Xu, Quan Wu, Qiang J Mol Cell Biol Articles The human genome contains millions of DNA regulatory elements and a large number of gene clusters, most of which have not been tested experimentally. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) programed with a synthetic single-guide RNA (sgRNA) emerges as a method for genome editing in virtually any organisms. Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs. Specifically, we found that, in cultured human cells and mice, efficient precise inversions of DNA fragments ranging in size from a few tens of bp to hundreds of kb could be generated. In addition, DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks (DSBs) on two homologous chromosomes (chromatids). Moreover, junctions of combinatorial inversions and duplications of the protocadherin (Pcdh) gene clusters induced by Cas9 with four sgRNAs could be detected. In mice, we obtained founders with alleles of precise inversions, duplications, and deletions of DNA fragments of variable sizes by CRISPR. Interestingly, we found that very efficient inversions were mediated by microhomology-mediated end joining (MMEJ) through short inverted repeats. We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice. Finally, we applied this CRISPR method to a regulatory element of the Pcdhα cluster and found a new role in the regulation of members of the Pcdhγ cluster. This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters. Oxford University Press 2015-08 2015-03-10 /pmc/articles/PMC4524425/ /pubmed/25757625 http://dx.doi.org/10.1093/jmcb/mjv016 Text en © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Articles
Li, Jinhuan
Shou, Jia
Guo, Ya
Tang, Yuanxiao
Wu, Yonghu
Jia, Zhilian
Zhai, Yanan
Chen, Zhifeng
Xu, Quan
Wu, Qiang
Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9
title Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9
title_full Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9
title_fullStr Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9
title_full_unstemmed Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9
title_short Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9
title_sort efficient inversions and duplications of mammalian regulatory dna elements and gene clusters by crispr/cas9
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524425/
https://www.ncbi.nlm.nih.gov/pubmed/25757625
http://dx.doi.org/10.1093/jmcb/mjv016
work_keys_str_mv AT lijinhuan efficientinversionsandduplicationsofmammalianregulatorydnaelementsandgeneclustersbycrisprcas9
AT shoujia efficientinversionsandduplicationsofmammalianregulatorydnaelementsandgeneclustersbycrisprcas9
AT guoya efficientinversionsandduplicationsofmammalianregulatorydnaelementsandgeneclustersbycrisprcas9
AT tangyuanxiao efficientinversionsandduplicationsofmammalianregulatorydnaelementsandgeneclustersbycrisprcas9
AT wuyonghu efficientinversionsandduplicationsofmammalianregulatorydnaelementsandgeneclustersbycrisprcas9
AT jiazhilian efficientinversionsandduplicationsofmammalianregulatorydnaelementsandgeneclustersbycrisprcas9
AT zhaiyanan efficientinversionsandduplicationsofmammalianregulatorydnaelementsandgeneclustersbycrisprcas9
AT chenzhifeng efficientinversionsandduplicationsofmammalianregulatorydnaelementsandgeneclustersbycrisprcas9
AT xuquan efficientinversionsandduplicationsofmammalianregulatorydnaelementsandgeneclustersbycrisprcas9
AT wuqiang efficientinversionsandduplicationsofmammalianregulatorydnaelementsandgeneclustersbycrisprcas9