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Large Genomic Fragment Deletions and Insertions in Mouse Using CRISPR/Cas9

ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. Howev...

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Autores principales: Zhang, Luqing, Jia, Ruirui, Palange, Norberto J., Satheka, Achim Cchitvsanzwhoh, Togo, Jacques, An, Yao, Humphrey, Mabwi, Ban, Luying, Ji, Yan, Jin, Honghong, Feng, Xuechao, Zheng, Yaowu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4372442/
https://www.ncbi.nlm.nih.gov/pubmed/25803037
http://dx.doi.org/10.1371/journal.pone.0120396
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author Zhang, Luqing
Jia, Ruirui
Palange, Norberto J.
Satheka, Achim Cchitvsanzwhoh
Togo, Jacques
An, Yao
Humphrey, Mabwi
Ban, Luying
Ji, Yan
Jin, Honghong
Feng, Xuechao
Zheng, Yaowu
author_facet Zhang, Luqing
Jia, Ruirui
Palange, Norberto J.
Satheka, Achim Cchitvsanzwhoh
Togo, Jacques
An, Yao
Humphrey, Mabwi
Ban, Luying
Ji, Yan
Jin, Honghong
Feng, Xuechao
Zheng, Yaowu
author_sort Zhang, Luqing
collection PubMed
description ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. However, the most mutations reported up to date are small insertions and deletions. In this report, CRISPR/Cas9 system was used to make large DNA fragment deletions and insertions, including entire Dip2a gene deletion, about 65kb in size, and β-galactosidase (lacZ) reporter gene insertion of larger than 5kb in mouse. About 11.8% (11/93) are positive for 65kb deletion from transfected and diluted ES clones. High targeting efficiencies in ES cells were also achieved with G418 selection, 46.2% (12/26) and 73.1% (19/26) for left and right arms respectively. Targeted large fragment deletion efficiency is about 21.4% of live pups or 6.0% of injected embryos. Targeted insertion of lacZ reporter with NEO cassette showed 27.1% (13/48) of targeting rate by ES cell transfection and 11.1% (2/18) by direct zygote injection. The procedures have bypassed in vitro transcription by directly co-injection of zygotes or co-transfection of embryonic stem cells with circular plasmid DNA. The methods are technically easy, time saving, and cost effective in generating mouse models and will certainly facilitate gene function studies.
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spelling pubmed-43724422015-04-04 Large Genomic Fragment Deletions and Insertions in Mouse Using CRISPR/Cas9 Zhang, Luqing Jia, Ruirui Palange, Norberto J. Satheka, Achim Cchitvsanzwhoh Togo, Jacques An, Yao Humphrey, Mabwi Ban, Luying Ji, Yan Jin, Honghong Feng, Xuechao Zheng, Yaowu PLoS One Research Article ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. However, the most mutations reported up to date are small insertions and deletions. In this report, CRISPR/Cas9 system was used to make large DNA fragment deletions and insertions, including entire Dip2a gene deletion, about 65kb in size, and β-galactosidase (lacZ) reporter gene insertion of larger than 5kb in mouse. About 11.8% (11/93) are positive for 65kb deletion from transfected and diluted ES clones. High targeting efficiencies in ES cells were also achieved with G418 selection, 46.2% (12/26) and 73.1% (19/26) for left and right arms respectively. Targeted large fragment deletion efficiency is about 21.4% of live pups or 6.0% of injected embryos. Targeted insertion of lacZ reporter with NEO cassette showed 27.1% (13/48) of targeting rate by ES cell transfection and 11.1% (2/18) by direct zygote injection. The procedures have bypassed in vitro transcription by directly co-injection of zygotes or co-transfection of embryonic stem cells with circular plasmid DNA. The methods are technically easy, time saving, and cost effective in generating mouse models and will certainly facilitate gene function studies. Public Library of Science 2015-03-24 /pmc/articles/PMC4372442/ /pubmed/25803037 http://dx.doi.org/10.1371/journal.pone.0120396 Text en © 2015 Zhang et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Zhang, Luqing
Jia, Ruirui
Palange, Norberto J.
Satheka, Achim Cchitvsanzwhoh
Togo, Jacques
An, Yao
Humphrey, Mabwi
Ban, Luying
Ji, Yan
Jin, Honghong
Feng, Xuechao
Zheng, Yaowu
Large Genomic Fragment Deletions and Insertions in Mouse Using CRISPR/Cas9
title Large Genomic Fragment Deletions and Insertions in Mouse Using CRISPR/Cas9
title_full Large Genomic Fragment Deletions and Insertions in Mouse Using CRISPR/Cas9
title_fullStr Large Genomic Fragment Deletions and Insertions in Mouse Using CRISPR/Cas9
title_full_unstemmed Large Genomic Fragment Deletions and Insertions in Mouse Using CRISPR/Cas9
title_short Large Genomic Fragment Deletions and Insertions in Mouse Using CRISPR/Cas9
title_sort large genomic fragment deletions and insertions in mouse using crispr/cas9
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4372442/
https://www.ncbi.nlm.nih.gov/pubmed/25803037
http://dx.doi.org/10.1371/journal.pone.0120396
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