Efficient CRISPR/Cas9 plasmids for rapid and versatile genome editing in Drosophila.
Bottom Line: The CRISPR-associated RNA-guided nuclease Cas9 has emerged as a powerful tool for genome engineering in a variety of organisms.To achieve efficient gene targeting rates in Drosophila, current approaches require either injection of in vitro transcribed RNAs or injection into transgenic Cas9-expressing embryos.Our strategy is independent of genetic background and does not require prior establishment of transgenic flies.
Affiliation: Institute of Molecular Biotechnology of the Austrian Academy of Sciences, 1030 Vienna, Austria.Show MeSH
Related in: MedlinePlus
Mentions: The human codon-optimized Streptococcus pyogenes Cas9D10A nickase (hSpCas9D10A) was cloned from pX335 (Cong et al. 2013) into the pHW Drosophila Gateway vector and the BbsI site in the SV40 3′-UTR mutated by PCR. The sgRNA expression cassette was then inserted into the NotI site of pHW-Cas9D10A by In-Fusion cloning (Clontech) of the U6:96Ab promoter amplified from genomic DNA and the chimeric sgRNA scaffold from pX335, resulting in plasmid pDCC1. To generate pDCC2, the N-terminus of Cas9D10A in pDCC1 was replaced with an AgeI-ApaI fragment of wild-type Cas9 from pX330 (Cong et al. 2013). Finally, we replaced the hsp70Ab promoter between the XbaI and HindIII sites in pDCC1 and pDCC2 with a 350-bp hsp70Bb promoter fragment to obtain plasmids pDCC5 and pDCC6, respectively (Figure 1A and Supporting Information, Figure S1).
Affiliation: Institute of Molecular Biotechnology of the Austrian Academy of Sciences, 1030 Vienna, Austria.