Using CRISPR-Cas9-based genome engineering tools in Drosophila melanogaster.

Abstract

Drosophila melanogaster has been used as a model organism for over a century. Mutant-based analyses have been used extensively to understand the genetic basis of different cellular processes, including development, neuronal function and diseases. Most of the earlier genetic mutants and specific tools were generated by random insertions and deletion strategies and then mapped to specific genomic loci. Since all genomic regions are not equally accessible to random mutations and insertions, many genes still remain uncharacterized. Low efficiency of targeted genomic manipulation approaches that rely on homologous recombination, and difficulty in generating resources for sequence-specific endonucleases, such as ZFNs (Zinc Finger Nucleases) and TALENs (Transcription Activator-Like Effector Nucleases), could not make these gene targeting techniques very popular. However, recently RNA directed DNA endonucleases, such as CRISPR-Cas, have transformed genome engineering owing to their comparative ease, versatility, and low expense. With the added advantage of preexisting genetic tools, CRISPR-Cas-based manipulations are being extensively used in Drosophila melanogaster and simultaneously being fine-tuned for specific experimental requirements. In this chapter, I will discuss various uses of CRISPR-Cas-based genetic engineering and specific design methods in Drosophila melanogaster. I will summarize various already available tools that are being utilized in conjunction with CRISPR-Cas technology to generate specific genetic manipulation and are being optimized to address specific questions. Finally, I will discuss the future directions of Drosophila genetics research and how CRISPR-Cas can be utilized to target specific questions, addressing which has not been possible thus far.