Farris M Heath, Texter Pamela A, Mora Agustin A, Wiles Michael V, Mac Garrigle Ellen F, Klaus Sybil A, Rosfjord Kristine
The MITRE Corporation, 7515 Colshire Drive, McLean, Virginia, 22102, USA.
The Jackson Laboratory, Technology Evaluation and Development, Bar Harbor, ME, USA.
BMC Genomics. 2020 Dec 2;21(1):856. doi: 10.1186/s12864-020-07233-2.
The 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.
In 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.
With 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.
用于基因组修饰的CRISPR技术的开发和应用正在迅速扩展。该领域的进展描述了经过策略性设计的新型CRISPR组件，以提高基因组序列内CRISPR编辑的精确性和可靠性。利用CRISPR组件靶向介导的诱导基因组断裂进行基因组修饰，借助同源定向修复（HDR）等细胞修复机制，以更高的精确性整合基因组编辑。
在本报告中，我们描述了在受CRISPR介导的使用HDR机制掺入供体DNA影响的典型低甲基化CpG岛（CGI）位置处甲基化的增加。通过使用全基因组亚硫酸氢盐测序对CpG甲基化模式进行表征，这些CGI甲基化破坏追踪了基因组编辑过程中供体DNA的插入。由同源定向重组介导的这些插入破坏了细胞内编辑的CGI及其在动物品系中的细胞谱系的世代甲基化模式稳定性，并代代持续。我们的方法描述了一种基于统计学的工作流程，用于指示修饰的CGI的位置，并提供了一种在CpG水平评估受影响CGI的甲基化组定向修饰的机制。
随着基因组修饰技术的进步，有必要检测基因组序列修饰对附带编辑的甲基化组所造成的甲基化变化的水平和持久性。体细胞或生殖细胞甲基化组的任何修饰都可能对在更敏感调控的基因组区域进行治疗性编辑时由CGI区域甲基化模式所控制的基因调控机制产生影响。这里描述的方法定位了在小鼠表观基因组中持续几代的定向修饰。虽然这种观察需要支持性的分子观察，如直接序列变化或基因表达变化，但表观遗传修饰的观察提供了一个指标，表明有意进行的基因组编辑在修饰后可能导致附带的、无意的表观基因组变化，并具有世代持久性。
