Abstract
BackgroundThe development and application of CRISPR technologies for the modification of the genome are rapidly expanding. Advances in the field describe new CRISPR components that are strategically engineered to improve the precision and reliability of CRISPR editing within the genome sequence. Genome modification using induced genome breaks that are targeted and mediated by CRISPR components leverage cellular mechanisms for repair like homology directed repair (HDR) to incorporate genomic edits with increased precision.ResultsIn this report, we describe the gain of methylation at typically hypomethylated CpG island (CGI) locations affected by the CRISPR-mediated incorporation of donor DNA using HDR mechanisms. With characterization of CpG methylation patterns using whole genome bisulfite sequencing, these CGI methylation disruptions trace the insertion of the donor DNA during the genomic edit. These insertions mediated by homology-directed recombination disrupt the generational methylation pattern stability of the edited CGI within the cells and their cellular lineage within the animal strain, persisting across generations. Our approach describes a statistically based workflow for indicating locations of modified CGIs and provides a mechanism for evaluating the directed modification of the methylome of the affected CGI at the CpG-level.ConclusionsWith advances in genome modification technology comes the need to detect the level and persistence of methylation change that modifications to the genomic sequence impose upon the collaterally edited methylome. Any modification of the methylome of somatic or germline cells could have implications for gene regulation mechanisms governed by the methylation patterns of CGI regions in the application of therapeutic edits of more sensitively regulated genomic regions. The method described here locates the directed modification of the mouse epigenome that persists over generations. While this observance would require supporting molecular observations such as direct sequence changes or gene expression changes, the observation of epigenetic modification provides an indicator that intentionally directed genomic edits can lead to collateral, unintentional epigenomic changes post modification with generational persistence.
Highlights
The development and application of CRISPR technologies for the modification of the genome are rapidly expanding
The epigenome modifications observed within the mouse strains were secondarily introduced into the genomes by homology-directed repair CRISPR edits
For the Cytosinephosphate-guanine site (CpG) locations of the defined CpG island (CGI) within the study, our evaluations focused on CGIs with an increased methylation profile at their CpG locations as compared to control animals, requiring a minimum of five bisulfite sequence calls at each CpG location for increased confidence in the downstream statistical assessments for determining and evaluating the edited CGIs
Summary
The development and application of CRISPR technologies for the modification of the genome are rapidly expanding. The emergence of rapid genome editing methodologies facilitated by CRISPR/Cas9 [1] and similar technologies has opened the ability to generated stable and precise edits within animal models [2], accelerating the rate of disease research and creating a shorter path toward disease treatments, therapeutic applications, and agricultural improvements. Tissue- and cell-targeted applications like somatic gene therapy can deliver genetic modification components to the respective targets via molecular and physical carriers, including liposomes [5,6,7], adeno-associated virus [8,9,10], lentivirus [11], and electroporation [12] These genome modification approaches have their respective challenges and limitations; these and other chemical and physical transfection methodologies have successfully generated representative mouse models of human disease, using CRISPR components. Many other species, including humans [13], have been genetically modified by CRISPR and CRISPR-like systems
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