Journal Article

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

Thelakkad Chathoth, Keerthi, Sanli, Ildem, Feil, Robert, Taylor, Gillian C., Kallimasioti-Pazi, Eirini M., Ballinger, Tracy, Wood, Andrew J., Meynert, Alison, Kelder, Martijn J. E., Lalevée, Sébastien

2018

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Alleles / Animals / Base Sequence / Bioinformatics / Biology and Life Sciences / Chromatin / Chromosome deletion / Chromosomes / Clonal deletion / CRISPR / CRISPR-Cas Systems - genetics / CRISPR-Cas Systems - physiology / Deoxyribonucleic acid / DNA / DNA Breaks, Double-Stranded / DNA methylation / DNA repair / DNA Repair - genetics / DNA Repair - physiology / DNA sequencing / Editing / Embryo cells / Endonucleases - metabolism / Epigenetics / Exposure / Gene Editing - methods / Gene expression / Genes / Genetics / Genome / Genome editing / Genomes / Heterochromatin / Heterochromatin - genetics / Heterochromatin - physiology / Heterogeneity / Homology / Human genetics / Influence / Kinetics / Life Sciences / Medicine / Methods / Mice / Mice, Inbred C57BL / Mouse Embryonic Stem Cells - physiology / Mutagenesis / Mutagenesis, Insertional / Mutagens / Mutation / Mutation - genetics / Nucleotide sequence / Nucleotide sequencing / Observations / Physiological aspects / Publishing / Recombinational DNA Repair - physiology / Repair / Research and Analysis Methods / Sequence Deletion / Short Reports / Software / Stem cell transplantation / Stem cells / Structure-function relationships / Supervision / Therapeutic applications