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CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences
CRISPR/Cas9 systems are a versatile tool for genome editing due to the highly efficient targeting of DNA sequences complementary to their RNA guide strands. However, it has been shown that RNA-guided Cas9 nuclease cleaves genomic DNA sequences containing mismatches to the guide strand. A better unde...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4066799/ https://www.ncbi.nlm.nih.gov/pubmed/24838573 http://dx.doi.org/10.1093/nar/gku402 |
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author | Lin, Yanni Cradick, Thomas J. Brown, Matthew T. Deshmukh, Harshavardhan Ranjan, Piyush Sarode, Neha Wile, Brian M. Vertino, Paula M. Stewart, Frank J. Bao, Gang |
author_facet | Lin, Yanni Cradick, Thomas J. Brown, Matthew T. Deshmukh, Harshavardhan Ranjan, Piyush Sarode, Neha Wile, Brian M. Vertino, Paula M. Stewart, Frank J. Bao, Gang |
author_sort | Lin, Yanni |
collection | PubMed |
description | CRISPR/Cas9 systems are a versatile tool for genome editing due to the highly efficient targeting of DNA sequences complementary to their RNA guide strands. However, it has been shown that RNA-guided Cas9 nuclease cleaves genomic DNA sequences containing mismatches to the guide strand. A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large mammalian genomes. Here we show that genomic sites could be cleaved by CRISPR/Cas9 systems when DNA sequences contain insertions (‘DNA bulge’) or deletions (‘RNA bulge’) compared to the RNA guide strand, and Cas9 nickases used for paired nicking can also tolerate bulges in one of the guide strands. Variants of single-guide RNAs (sgRNAs) for four endogenous loci were used as model systems, and their cleavage activities were quantified at different positions with 1- to 5-bp bulges. We further investigated 114 putative genomic off-target loci of 27 different sgRNAs and confirmed 15 off-target sites, each harboring a single-base bulge and one to three mismatches to the guide strand. Our results strongly indicate the need to perform comprehensive off-target analysis related to DNA and sgRNA bulges in addition to base mismatches, and suggest specific guidelines for reducing potential off-target cleavage. |
format | Online Article Text |
id | pubmed-4066799 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-40667992014-06-24 CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences Lin, Yanni Cradick, Thomas J. Brown, Matthew T. Deshmukh, Harshavardhan Ranjan, Piyush Sarode, Neha Wile, Brian M. Vertino, Paula M. Stewart, Frank J. Bao, Gang Nucleic Acids Res Synthetic Biology and Chemistry CRISPR/Cas9 systems are a versatile tool for genome editing due to the highly efficient targeting of DNA sequences complementary to their RNA guide strands. However, it has been shown that RNA-guided Cas9 nuclease cleaves genomic DNA sequences containing mismatches to the guide strand. A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large mammalian genomes. Here we show that genomic sites could be cleaved by CRISPR/Cas9 systems when DNA sequences contain insertions (‘DNA bulge’) or deletions (‘RNA bulge’) compared to the RNA guide strand, and Cas9 nickases used for paired nicking can also tolerate bulges in one of the guide strands. Variants of single-guide RNAs (sgRNAs) for four endogenous loci were used as model systems, and their cleavage activities were quantified at different positions with 1- to 5-bp bulges. We further investigated 114 putative genomic off-target loci of 27 different sgRNAs and confirmed 15 off-target sites, each harboring a single-base bulge and one to three mismatches to the guide strand. Our results strongly indicate the need to perform comprehensive off-target analysis related to DNA and sgRNA bulges in addition to base mismatches, and suggest specific guidelines for reducing potential off-target cleavage. Oxford University Press 2014-07-01 2014-05-16 /pmc/articles/PMC4066799/ /pubmed/24838573 http://dx.doi.org/10.1093/nar/gku402 Text en © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), 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 | Synthetic Biology and Chemistry Lin, Yanni Cradick, Thomas J. Brown, Matthew T. Deshmukh, Harshavardhan Ranjan, Piyush Sarode, Neha Wile, Brian M. Vertino, Paula M. Stewart, Frank J. Bao, Gang CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences |
title | CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences |
title_full | CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences |
title_fullStr | CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences |
title_full_unstemmed | CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences |
title_short | CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences |
title_sort | crispr/cas9 systems have off-target activity with insertions or deletions between target dna and guide rna sequences |
topic | Synthetic Biology and Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4066799/ https://www.ncbi.nlm.nih.gov/pubmed/24838573 http://dx.doi.org/10.1093/nar/gku402 |
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