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Efficient CRISPR/Cas9 plasmids for rapid and versatile genome editing in Drosophila.

Gokcezade J, Sienski G, Duchek P - G3 (Bethesda) (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biotechnology of the Austrian Academy of Sciences, 1030 Vienna, Austria.

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A bicistronic Drosophila CRISPR/Cas9 vector. (A) Schematic map of pDCC6, with the sgRNA cassette under the control of the Drosophila U6:96Ab (U6-2) promoter as well as an hsp70Bb promoter driving Cas9 expression. (B) gRNA sequences are inserted between the U6 promoter and the sgRNA scaffold via two BbsI sites. (C) gRNAs are cloned as complementary oligonucleotide pairs with suitable overhangs and an additional G (bold, required for RNA PolIII transcription) preceding the target-specific 20 nt protospacer (gray).
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fig1: A bicistronic Drosophila CRISPR/Cas9 vector. (A) Schematic map of pDCC6, with the sgRNA cassette under the control of the Drosophila U6:96Ab (U6-2) promoter as well as an hsp70Bb promoter driving Cas9 expression. (B) gRNA sequences are inserted between the U6 promoter and the sgRNA scaffold via two BbsI sites. (C) gRNAs are cloned as complementary oligonucleotide pairs with suitable overhangs and an additional G (bold, required for RNA PolIII transcription) preceding the target-specific 20 nt protospacer (gray).

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).


Efficient CRISPR/Cas9 plasmids for rapid and versatile genome editing in Drosophila.

Gokcezade J, Sienski G, Duchek P - G3 (Bethesda) (2014)

A bicistronic Drosophila CRISPR/Cas9 vector. (A) Schematic map of pDCC6, with the sgRNA cassette under the control of the Drosophila U6:96Ab (U6-2) promoter as well as an hsp70Bb promoter driving Cas9 expression. (B) gRNA sequences are inserted between the U6 promoter and the sgRNA scaffold via two BbsI sites. (C) gRNAs are cloned as complementary oligonucleotide pairs with suitable overhangs and an additional G (bold, required for RNA PolIII transcription) preceding the target-specific 20 nt protospacer (gray).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232553&req=5

fig1: A bicistronic Drosophila CRISPR/Cas9 vector. (A) Schematic map of pDCC6, with the sgRNA cassette under the control of the Drosophila U6:96Ab (U6-2) promoter as well as an hsp70Bb promoter driving Cas9 expression. (B) gRNA sequences are inserted between the U6 promoter and the sgRNA scaffold via two BbsI sites. (C) gRNAs are cloned as complementary oligonucleotide pairs with suitable overhangs and an additional G (bold, required for RNA PolIII transcription) preceding the target-specific 20 nt protospacer (gray).
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biotechnology of the Austrian Academy of Sciences, 1030 Vienna, Austria.

Show MeSH
Related in: MedlinePlus