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In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining

The CRISPR/Cas9 system facilitates precise DNA modifications by generating RNA-guided blunt-ended double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called...

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Autores principales: Geisinger, Jonathan M., Turan, Sören, Hernandez, Sophia, Spector, Laura P., Calos, Michele P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856974/
https://www.ncbi.nlm.nih.gov/pubmed/26762978
http://dx.doi.org/10.1093/nar/gkv1542
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author Geisinger, Jonathan M.
Turan, Sören
Hernandez, Sophia
Spector, Laura P.
Calos, Michele P.
author_facet Geisinger, Jonathan M.
Turan, Sören
Hernandez, Sophia
Spector, Laura P.
Calos, Michele P.
author_sort Geisinger, Jonathan M.
collection PubMed
description The CRISPR/Cas9 system facilitates precise DNA modifications by generating RNA-guided blunt-ended double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting these breaks to insert exogenous PCR-generated sequences in a homology-independent manner without loss of additional nucleotides. This method is useful for making precise additions to the genome such as insertions of marker gene cassettes or functional elements, without the need for homology arms. We successfully utilized this method in human and mouse cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 36% in HEK293 cells without selection. We also created versions of Cas9 fused to the FKBP12-L106P destabilization domain in an effort to improve Cas9 performance. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches.
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spelling pubmed-48569742016-05-09 In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining Geisinger, Jonathan M. Turan, Sören Hernandez, Sophia Spector, Laura P. Calos, Michele P. Nucleic Acids Res Methods Online The CRISPR/Cas9 system facilitates precise DNA modifications by generating RNA-guided blunt-ended double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting these breaks to insert exogenous PCR-generated sequences in a homology-independent manner without loss of additional nucleotides. This method is useful for making precise additions to the genome such as insertions of marker gene cassettes or functional elements, without the need for homology arms. We successfully utilized this method in human and mouse cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 36% in HEK293 cells without selection. We also created versions of Cas9 fused to the FKBP12-L106P destabilization domain in an effort to improve Cas9 performance. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches. Oxford University Press 2016-05-05 2016-01-13 /pmc/articles/PMC4856974/ /pubmed/26762978 http://dx.doi.org/10.1093/nar/gkv1542 Text en © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Methods Online
Geisinger, Jonathan M.
Turan, Sören
Hernandez, Sophia
Spector, Laura P.
Calos, Michele P.
In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
title In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
title_full In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
title_fullStr In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
title_full_unstemmed In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
title_short In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
title_sort in vivo blunt-end cloning through crispr/cas9-facilitated non-homologous end-joining
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856974/
https://www.ncbi.nlm.nih.gov/pubmed/26762978
http://dx.doi.org/10.1093/nar/gkv1542
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