Heat shock-optimized CRISPR/Cas9 system for visible clonal analysis and mutant generation in Drosophila.

In Drosophila genetic studies, clonal analysis such as mosaic and Mosaic Analysis with a Repressible Cell Marker has been widely used to investigate gene function. Recently, the CRISPR/Cas9 system has been established as a powerful tool for efficient mutant generation; however, its application in clonal analysis has been rarely reported. Here, we present a suite of Gal4/UAS-Cas9 binary expression systems that integrate UAS-Cas9 and multiple-sgRNAs (single-guide RNAs) into a single plasmid. These systems facilitate versatile applications, enabling Gal4-driven direct phenotypic studies, approximate clonal analysis, in vitro cell transfection, and stable mutant generation, among which, the third-generation constructs: G3a/b incorporate visible labeling strategies for marking approximate clonal regions. In addition, compared to continuously active drivers, we found that the short-pulse-induced heat shock-Gal4 (hs-Gal4) was sufficient to induce high clonal efficiency and generate larger clones. In the germline, short-pulse heat shock is also effective. It reduces residual Cas9 activity in the germline stem cells, thereby minimizing the risk of affecting germline stem cell survival and improving mutant acquisition.