Abstract：
CRISPR-Cas greatly facilitated the integration of exogenous sequences into specific loci. However, knockin generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Here, we describe SEED/Harvest, a method to generate knockins in Drosophila, based on CRISPR-Cas and the single-strand annealing (SSA) repair pathway. In SEED (from \"scarless editing by element deletion\"), a switchable cassette is first integrated into the target locus. In a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knockin generation of both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatiotemporal control of protein labeling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knockins, we developed a genetic toolbox by functionalizing the ALFA nanobody.
摘要：
CRISPR-Cas极大地促进了外源序列整合到特定基因座中。然而,在多细胞动物中敲入代仍然具有挑战性，部分是由于插入筛选的复杂性。这里,我们描述了种子/收获，一种在果蝇中产生敲击蛋白的方法，基于CRISPR-Cas和单链退火(SSA)修复途径。在SEED中（来自“通过元素删除进行无疤痕编辑”），首先将可切换盒整合到目标基因座中。在随后的CRISPR触发的修复事件中，由SSA解决，磁带被无缝地移除。SEED盒的种系切除允许串联的荧光蛋白和短蛋白标签的快速和稳健的敲入生成。Cas9的组织特异性表达导致体细胞盒切除，赋予蛋白质标记的时空控制和突变体的条件拯救。最后,为了实现条件蛋白标记和短标签敲击素的操作，我们通过功能化ALFA纳米抗体开发了一个基因工具箱。
