Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair

被引:70
作者
Kallimasioti-Pazi, Eirini M. [1 ]
Chathoth, Keerthi Thelakkad [1 ,4 ]
Taylor, Gillian C. [1 ]
Meynert, Alison [1 ]
Ballinger, Tracy [1 ]
Kelder, Martijn J. E. [1 ]
Lalevee, Sebastien [2 ,3 ,5 ]
Sanli, Ildem [2 ,3 ,6 ]
Feil, Robert [2 ,3 ]
Wood, Andrew J. [1 ]
机构
[1] Univ Edinburgh, Inst Genet & Mol Med, MRC Human Genet Unit, Edinburgh, Midlothian, Scotland
[2] CNRS, Inst Mol Genet Montpellier IGMM, Montpellier, France
[3] Univ Montpellier, Montpellier, France
[4] Univ Essex, Sch Biol Sci, Colchester, Essex, England
[5] 4 Antibody AG, Basel, Switzerland
[6] Inst Pasteur, Dept Dev & Stem Cell Biol, Paris, France
基金
英国惠康基金; 英国医学研究理事会;
关键词
READ ALIGNMENT; GENOMIC DNA; CHROMATIN; DYNAMICS; BINDING; CAS9; RNA; REGIONS; IMPACT; BASE;
D O I
10.1371/journal.pbio.2005595
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem in cycling mouse embryonic stem cells (mESCs). Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of mutations induced by CRISPR-Cas9 can be unequivocally attributed to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. In contrast, the outcome of mutagenic DNA repair was unaffected by chromatin state, with similar efficiencies of homology-directed repair (HDR) and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint, of CRISPR-Cas9 genome editing and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.
引用
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页数:22
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