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Detection of CRISPR-mediated genome modifications through altered methylation patterns of CpG islands
BMC Genomics ( IF 4.4 ) Pub Date : 2020-12-02 , DOI: 10.1186/s12864-020-07233-2 M. Heath Farris , Pamela A. Texter , Agustin A. Mora , Michael V. Wiles , Ellen F. Mac Garrigle , Sybil A. Klaus , Kristine Rosfjord
BMC Genomics ( IF 4.4 ) Pub Date : 2020-12-02 , DOI: 10.1186/s12864-020-07233-2 M. Heath Farris , Pamela A. Texter , Agustin A. Mora , Michael V. Wiles , Ellen F. Mac Garrigle , Sybil A. Klaus , Kristine Rosfjord
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.
中文翻译:
通过改变CpG岛的甲基化模式来检测CRISPR介导的基因组修饰
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新的CRISPR组件,这些组件经过战略性设计以提高基因组序列中CRISPR编辑的精度和可靠性。使用由CRISPR组件靶向和介导的诱导基因组断裂进行基因组修饰,利用细胞机制进行修复,如同源性定向修复(HDR),以更高的精度整合基因组编辑。在本报告中,我们描述了在典型的低甲基化CpG岛(CGI)位置,使用HDR机制受CRISPR介导的供体DNA掺入的影响,甲基化的获得。通过使用全基因组亚硫酸氢盐测序来表征CpG甲基化模式,这些CGI甲基化破坏可追踪基因组编辑过程中供体DNA的插入。由同源性指导的重组介导的这些插入破坏了细胞内编辑的CGI及其动物谱系中细胞谱系的世代甲基化模式稳定性,持续了几代人。我们的方法描述了一种基于统计的工作流,用于指示修饰的CGI的位置,并提供了一种机制,用于在CpG级别评估受影响CGI的甲基化组的定向修饰。随着基因组修饰技术的进步,需要检测甲基化变化的水平和持久性,所述甲基化变化是对基因组序列的修饰强加于附带编辑的甲基化组上的。体细胞或种系细胞甲基化组的任何修饰都可能对由CGI区的甲基化模式控制的基因调控机制产生影响,在更敏感调控的基因组区域的治疗编辑中应用。这里描述的方法定位了小鼠表观基因组的定向修饰,该修饰持续了数代之久。尽管这种观察需要支持分子观察,例如直接序列改变或基因表达改变,但是表观遗传修饰的观察提供了这样的指示,即有意指导的基因组编辑可以在修饰后代产生持久的附带的,无意的表观基因组改变。
更新日期:2020-12-02
中文翻译:
通过改变CpG岛的甲基化模式来检测CRISPR介导的基因组修饰
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新的CRISPR组件,这些组件经过战略性设计以提高基因组序列中CRISPR编辑的精度和可靠性。使用由CRISPR组件靶向和介导的诱导基因组断裂进行基因组修饰,利用细胞机制进行修复,如同源性定向修复(HDR),以更高的精度整合基因组编辑。在本报告中,我们描述了在典型的低甲基化CpG岛(CGI)位置,使用HDR机制受CRISPR介导的供体DNA掺入的影响,甲基化的获得。通过使用全基因组亚硫酸氢盐测序来表征CpG甲基化模式,这些CGI甲基化破坏可追踪基因组编辑过程中供体DNA的插入。由同源性指导的重组介导的这些插入破坏了细胞内编辑的CGI及其动物谱系中细胞谱系的世代甲基化模式稳定性,持续了几代人。我们的方法描述了一种基于统计的工作流,用于指示修饰的CGI的位置,并提供了一种机制,用于在CpG级别评估受影响CGI的甲基化组的定向修饰。随着基因组修饰技术的进步,需要检测甲基化变化的水平和持久性,所述甲基化变化是对基因组序列的修饰强加于附带编辑的甲基化组上的。体细胞或种系细胞甲基化组的任何修饰都可能对由CGI区的甲基化模式控制的基因调控机制产生影响,在更敏感调控的基因组区域的治疗编辑中应用。这里描述的方法定位了小鼠表观基因组的定向修饰,该修饰持续了数代之久。尽管这种观察需要支持分子观察,例如直接序列改变或基因表达改变,但是表观遗传修饰的观察提供了这样的指示,即有意指导的基因组编辑可以在修饰后代产生持久的附带的,无意的表观基因组改变。