Abstract:

The typeⅡ CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated protein 9) is an efficient RNA-guided genome-editing technique. Guided by sgRNA, the Cas9 endonuclease generates site-specific double-stranded breaks (DSB) at specific site, which is amenable to repair by homology-directed repair (HDR) to generate a designed knock-out or knock-in transgene. In combination with CRISPR/Cas9 and Cre/loxP or FLP/FRT system, efficient gene targeting can be achieved, and meanwhile screening markers introduced can be readily removed except a 34-base pair residual fragment. Thus, difficulties remain in accurate editing of the genome without introducing any extraneous sequences. In human induced pluripotent stem cells (iPSCs), a two-step strategy has been developed using CRISPR/Cas9 and the piggyBac system to establish a seamless genomic editing, in which CRISPR/Cas9 is initially used to introduce mutations along with screening markers by HDR, then the markers are precisely excised by piggyBac transposase. Using this strategy, we have successfully transformed the tyrosine to cysteine at position 21 within the 18th exon of the CG4894 gene in the Drosophila genome without introducing any extraneous sequence. Hence, this strategy provides more options for precise and seamless editing of the Drosophila genome.

Key words: Drosophila, seamless genome editing, CRISPR/Cas9, piggyBac