CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.
Bottom Line: Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila.Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency.Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner.
Affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.Show MeSH
Related in: MedlinePlus
Mentions: We also targeted the cid (cenH3) gene, which encodes a protein essential for somatic centromere assembly and embryogenesis (Blower and Karpen 2001; Blower et al. 2006). We induced 10UAS-Cas9 expression using the nos-Gal4 driver to detect phenotypes in the testis. Testis size was greatly reduced (Figure 4A), probably because nos-Gal4 is expressed very early during spermatogenesis (Rørth 1998). Disruption of the cid gene during early spermatogenesis led to a failure in cell division and to male sterility. We could not detect any mature sperm in the seminal vesicles of the mutated testes (Figure 4A), likely explaining the observed sterility. Low temperature−induced (25°) conditional mutagenesis using both promoters resulted in a small fraction of flies with defects in testis tissue (Figure 4B). However, induced mutagenesis at 28° resulted in a high fraction of flies with defects for both promoters. Notably, all examined mutated male flies under the control of the CR7T promoter (34 of 34) were 100% sterile and showed severe phenotypes in the testes at 28° (Figure 4B).
Affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.