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DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA
To investigate whether and how CRISPR-Cas9 on-target and off-target activities are affected by chromatin in eukaryotic cells, we first identified a series of identical endogenous DNA sequences present in both open and closed chromatin regions and then measured mutation frequencies at these sites in...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280750/ https://www.ncbi.nlm.nih.gov/pubmed/30413470 http://dx.doi.org/10.1101/gr.236620.118 |
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author | Kim, Daesik Kim, Jin-Soo |
author_facet | Kim, Daesik Kim, Jin-Soo |
author_sort | Kim, Daesik |
collection | PubMed |
description | To investigate whether and how CRISPR-Cas9 on-target and off-target activities are affected by chromatin in eukaryotic cells, we first identified a series of identical endogenous DNA sequences present in both open and closed chromatin regions and then measured mutation frequencies at these sites in human cells using Cas9 complexed with matched or mismatched sgRNAs. Unlike matched sgRNAs, mismatched sgRNAs were highly sensitive to chromatin states, suggesting that off-target but not on-target DNA cleavage is hindered by chromatin. We next performed Digenome-seq using cell-free chromatin DNA (now termed DIG-seq) and histone-free genomic DNA in parallel and found that only a subset of sites, cleaved in histone-free DNA, were cut in chromatin DNA, suggesting that chromatin can inhibit Cas9 off-target effects in favor of its genome-wide specificity in cells. |
format | Online Article Text |
id | pubmed-6280750 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-62807502018-12-26 DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA Kim, Daesik Kim, Jin-Soo Genome Res Method To investigate whether and how CRISPR-Cas9 on-target and off-target activities are affected by chromatin in eukaryotic cells, we first identified a series of identical endogenous DNA sequences present in both open and closed chromatin regions and then measured mutation frequencies at these sites in human cells using Cas9 complexed with matched or mismatched sgRNAs. Unlike matched sgRNAs, mismatched sgRNAs were highly sensitive to chromatin states, suggesting that off-target but not on-target DNA cleavage is hindered by chromatin. We next performed Digenome-seq using cell-free chromatin DNA (now termed DIG-seq) and histone-free genomic DNA in parallel and found that only a subset of sites, cleaved in histone-free DNA, were cut in chromatin DNA, suggesting that chromatin can inhibit Cas9 off-target effects in favor of its genome-wide specificity in cells. Cold Spring Harbor Laboratory Press 2018-12 /pmc/articles/PMC6280750/ /pubmed/30413470 http://dx.doi.org/10.1101/gr.236620.118 Text en © 2018 Kim and Kim; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by-nc/4.0/ This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. |
spellingShingle | Method Kim, Daesik Kim, Jin-Soo DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA |
title | DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA |
title_full | DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA |
title_fullStr | DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA |
title_full_unstemmed | DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA |
title_short | DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA |
title_sort | dig-seq: a genome-wide crispr off-target profiling method using chromatin dna |
topic | Method |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280750/ https://www.ncbi.nlm.nih.gov/pubmed/30413470 http://dx.doi.org/10.1101/gr.236620.118 |
work_keys_str_mv | AT kimdaesik digseqagenomewidecrisprofftargetprofilingmethodusingchromatindna AT kimjinsoo digseqagenomewidecrisprofftargetprofilingmethodusingchromatindna |