Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters inDrosophila

Abstract

The flySAM/CRISPRa system has recently emerged as a powerful tool for gain-of-function studies in Drosophilamelanogaster. 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.

Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells

“…As the Drosophila genome is relatively compact, one concern for CRISPRa is the collateral activation of adjacent genes. Previous work has shown that sgRNAs targeting areas beyond –600 bp from the TSS in Drosophila lose efficiency ( Mao et al, 2020 ), which may alleviate the concern of collateral activation when the sgRNAs target sites are over 600 bp away from the TSS of neighboring genes. In some cases, sgRNAs are designed within both promoters of closely spaced divergent genes.…”