利用染色质状态信息提高小麦基因编辑靶位点预测精确性

Gene editing has become a powerful tool for global agriculture, providing important molecular means for further improving crop yield, resistance and boosting nutrition efficiency. Recent studies have found that nucleosome entanglement with DNA prevents the Cas9 / sgRNA complex from binding to the target site, thereby reducing the editing efficiency of the CRISPR / Cas9 system. Wheat is one of the most important staple crops in the world. Because it is a large allotroic hexaploid genome, it needs to edit multiple copies at the same time, and the genome has a high degree of DNA methylation, resulting in inefficiency in gene editing. Based on our established wheat D genome methylation analysis and high-throughput computational biology analysis, this project uses the techniques of machine learning and artificial intelligence, combined with high-throughput sequencing and data from public databases to generate chromatin State of high-precision prediction to study the impact of chromosome status on gene editing efficiency, and by constructing CRISPR / Cas9 transient expression mixed pool library method to predict the results of validation combined with experimental data to further improve the model. This study will improve the gene editing efficiency of CRISPR / Cas9 system, provide the scientific basis for further study on the molecular mechanism of CRISPR / Cas9, and provide the basis and clues for the frontier research on the chromatin modification nucleosome structure of wheat.

基因编辑技术已成为未来改变全球农业的强大工具，为进一步提高作物产量，抗逆性，提高营养效率等提供重要的分子手段.研究发现核小体缠绕DNA会阻碍Cas9/sgRNA复合体结合靶位点，从而降低CRISPR/Cas9系统的编辑效率。小麦是世界重要的粮食作物之一，因其为基因组巨大的异源六倍体，需要同时对多个拷贝进行定向编辑，并且基因组甲基化程度高，导致小麦基因组编辑效率低下。本申请项目在我们已建立的小麦D基因组甲基化分析和高通量计算生物学分析的基础上，利用机器学习结合高通量测序以及公共数据库中数据实现对染色质状态的高精度预测，研究染色体状态对基因编辑效率的影响，并通过构建CRISPR/Cas9瞬时表达混池文库的方法对预测结果进行验证并结合实验数据对模型进一步提高。本研究将提高CRISPR/Cas9系统基因编辑效率，为深入研究小麦中CRISPR/Cas9作用分子机制提供基础和线索

本文主要研究组蛋白去乙酰化酶抑制剂（HDACi）与染色质状态以及CRISPR/Cas9 的编辑效率之间的关系。利用不同浓度的尼克酰胺和丁酸钠对小麦幼苗处理，结果显示丁酸钠处理会抑制幼苗的生长，而尼克酰胺对幼苗影响较小。因此，后续尼克酰胺被用来处理含有Gus，TaWaxy以及TaAgo4a基因编辑载体但是没有发生突变的转基因植株的幼胚，结果发现尼克酰胺处理提高了小麦的编辑效率最高可以从0%提高到36%。同时，对含有TaWaxy基因编辑载体而且没有发生突变的转基因植株的成熟胚进行了尼克酰胺处理，基因编辑效率也从0%提高到了13.3%。此外，我们在转基因中添加尼克酰胺，将单碱基编辑效率提高了3倍左右。这些结果都证明尼克酰胺处理能提高小麦的基因编辑效率。