Research Center for Infectious Diseases, University of Würzburg, 97080 Würzburg, Germany.
Department of Veterinary Sciences, Experimental Parasitology, Ludwig-Maximilians-Universität München, 80752 Munich, Germany.
Nucleic Acids Res. 2018 Oct 12;46(18):e106. doi: 10.1093/nar/gky517.
Despite their importance for most DNA-templated processes, the function of individual histone modifications has remained largely unknown because in vivo mutational analyses are lacking. The reason for this is that histone genes are encoded by multigene families and that tools to simultaneously edit multiple genomic loci with high efficiency are only now becoming available. To overcome these challenges, we have taken advantage of the power of CRISPR-Cas9 for precise genome editing and of the fact that most DNA repair in the protozoan parasite Trypanosoma brucei occurs via homologous recombination. By establishing an episome-based CRISPR-Cas9 system for T. brucei, we have edited wild type cells without inserting selectable markers, inserted a GFP tag between an ORF and its 3'UTR, deleted both alleles of a gene in a single transfection, and performed precise editing of genes that exist in multicopy arrays, replacing histone H4K4 with H4R4 in the absence of detectable off-target effects. The newly established genome editing toolbox allows for the generation of precise mutants without needing to change other regions of the genome, opening up opportunities to study the role of individual histone modifications, catalytic sites of enzymes or the regulatory potential of UTRs in their endogenous environments.
尽管单个组蛋白修饰对于大多数 DNA 模板过程都很重要，但由于缺乏体内突变分析，其功能在很大程度上仍是未知的。造成这种情况的原因是，组蛋白基因是由多基因家族编码的，并且能够高效地同时编辑多个基因组位点的工具现在才刚刚出现。为了克服这些挑战，我们利用了 CRISPR-Cas9 进行精确基因组编辑的能力，以及原生动物寄生虫锥虫中大多数 DNA 修复都是通过同源重组发生的这一事实。通过为锥虫建立基于附加体的 CRISPR-Cas9 系统，我们在不插入选择性标记的情况下编辑了野生型细胞，在一个 ORF 和其 3'UTR 之间插入了 GFP 标签，在单次转染中删除了一个基因的两个等位基因，并对存在于多拷贝阵列中的基因进行了精确编辑，在没有可检测的脱靶效应的情况下用 H4R4 取代了 H4K4。新建立的基因组编辑工具箱允许生成无需改变基因组其他区域的精确突变体，为研究单个组蛋白修饰、酶的催化位点或 UTR 的调节潜力在其内源环境中的作用提供了机会。
