Strategies for Efficient Genome Editing Using CRISPR-Cas9
Strategies for Efficient Genome Editing Using CRISPR-Cas9
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DOI：
10.1534/genetics.118.301775
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发表时间：
2019-02-01
期刊：
影响因子：
3.3
通讯作者：
Meyer, Barbara J.
中科院分区：
文献类型：
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作者：
Farboud, Behnom;Severson, Aaron F.;Meyer, Barbara J.
靶向DNA内切核酸酶CRISPR-Cas9通过在模型和非模型生物中实现强大的基因组编辑，改变了生物过程的分析。尽管通过指导RNA将Cas9引导至其靶DNA的规则是简单的，但对于不精确的易错修复和精确的模板化修复，编辑效率和修复结果存在很大差异。我们发现，从双链断裂（DSB）的不精确和精确的DNA修复是不对称的，有利于修复在一个方向。利用这些知识，我们设计了RNA指导和修复模板，增加了不精确插入和缺失的频率，并大大提高了秀丽隐杆线虫中点突变的精确插入。我们还设计了策略，插入长（10 kb）的外源序列，并纳入多个核苷酸取代在一个相当大的距离DSB。我们扩展了适合不同线虫物种的共转换标记库。这些选择性标记使得能够快速鉴定Cas9编辑的动物，这些动物也可能在所需靶标中携带编辑。最后，我们通过开发具有等位基因特异性Cas9靶标的基因座来探索DSB修复事件的时间、位置、频率、性别依赖性和类别，所述等位基因特异性Cas9靶标可以在来自雄性或雌雄同体生殖细胞的交配期间贡献。我们发现了母系和父系贡献的基因组之间编辑效率的显著差异。此外，不精确的修复和精确的修复外源修复模板发生高频率受精前后。我们的策略提高了Cas9靶向效率，深入了解DSB修复的时机和机制，并建立了实现可预测的高频率精确和不精确修复结果的指导方针。
The targetable DNA endonuclease CRISPR-Cas9 has transformed analysis of biological processes by enabling robust genome editing in model and nonmodel organisms. Although rules directing Cas9 to its target DNA via a guide RNA are straightforward, wide variation occurs in editing efficiency and repair outcomes for both imprecise error-prone repair and precise templated repair. We found that imprecise and precise DNA repair from double-strand breaks (DSBs) is asymmetric, favoring repair in one direction. Using this knowledge, we designed RNA guides and repair templates that increased the frequency of imprecise insertions and deletions and greatly enhanced precise insertion of point mutations in Caenorhabditis elegans. We also devised strategies to insert long (10 kb) exogenous sequences and incorporate multiple nucleotide substitutions at a considerable distance from DSBs. We expanded the repertoire of co-conversion markers appropriate for diverse nematode species. These selectable markers enable rapid identification of Cas9-edited animals also likely to carry edits in desired targets. Lastly, we explored the timing, location, frequency, sex dependence, and categories of DSB repair events by developing loci with allele-specific Cas9 targets that can be contributed during mating from either male or hermaphrodite germ cells. We found a striking difference in editing efficiency between maternally and paternally contributed genomes. Furthermore, imprecise repair and precise repair from exogenous repair templates occur with high frequency before and after fertilization. Our strategies enhance Cas9-targeting efficiency, lend insight into the timing and mechanisms of DSB repair, and establish guidelines for achieving predictable precise and imprecise repair outcomes with high frequency.