The flySAM/CRISPRa system has recently emerged as a powerful tool for gain-of-function studies in Drosophila melanogaster. This system includes Gal4/UAS-driven dCas9 activators and U6 promoter-controlled sgRNA. Having established dCas9 activators superior to other combinations, to further enhance the efficiency of the targeting activators we systematically optimized the parameters of the sgRNA. Interestingly, the most efficient sgRNAs were found to accumulate in the region from -150bp to -450bp upstream of the transcription start site (TSS), and the activation efficiency showed a strong positive correlation with the GC content of the sgRNA targeting sequence. In addition, the target region is dominant to the GC content, as sgRNAs targeting areas beyond -600bp from the TSS lose efficiency even when containing 75% GC. Surprisingly, when comparing the activities of sgRNAs targeting to either DNA strand, sgRNAs targeting to the non-template strand outperform those complementary to the template strand, both in cells and in vivo. In summary, we define criteria for sgRNA design which will greatly facilitate the application of CRISPRa in gain-of-function studies.

FlySAM/CRISPRa 系统最近已成为果蝇功能获得研究的强大工具。该系统包括 Gal4/UAS 驱动的 dCas9 激活剂和 U6 启动子控制的 sgRNA。在建立了优于其他组合的 dCas9 激活剂后，为了进一步提高靶向激活剂的效率，我们系统地优化了 sgRNA 的参数。有趣的是，我们发现最有效的sgRNA聚集在转录起始位点（TSS）上游-150bp至-450bp的区域，并且激活效率与sgRNA靶向序列的GC含量呈强正相关。此外，目标区域对 GC 含量占主导地位，因为即使含有 75% GC，目标区域超过 TSS -600bp 的 sgRNA 也会失去效率。令人惊讶的是，当比较靶向任一 DNA 链的 sgRNA 的活性时，无论是在细胞中还是在体内，针对非模板链的 sgRNA 均优于与模板链互补的 sgRNA。总之，我们定义了 sgRNA 设计标准，这将极大地促进 CRISPRa 在功能获得研究中的应用。