Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells.
Bottom Line: Homology arms as short as 29 nt appended to the PCR primer resulted in detectable integration, slightly longer extensions are beneficial.We confirmed established rules for S. pyogenes cas9 sgRNA design and demonstrate that the complementarity region allows length variation and 5'-extensions.The system can be used for epitope tagging or reporter gene knock-ins in an experimental setup that can in principle be fully automated.
Affiliation: Gene Center and Dept. of Biochemistry, Ludwig-Maximilians-Universität, Feodor-Lynen-Str. 25, D-81377 München, Germany.Show MeSH
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Mentions: To generate sgRNA templates for in vitro transcription via PCR, the oligonucleotide 5′-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC-3′ served as PCR template with a sense primer containing the T7 promoter and targeting sequence (e.g. 5′-taatacgactcactataGCCTAGGGATAACAGGGTAATGgttttagagct-3′) and 5′-GCACCGACTCGGTGCCACT-3′ as antisense primer. For the 3′-extended CRISPR's, oligo 5′-GTGAGCAAGGGCGAGGAGgcaccgactcggtgccact-3′ served as antisense PCR primer. Details on CRISPR target site oligonucleotides employed are given in Figure 2A and the supplementary information. In vitro transcription was performed as previously described for dsRNA (70), the sgRNA products were purified via a Qiagen PCR purification kit. PCR amplification of the U6-C promoter with a T7-extension was achieved with oligonucleotides 5′-GCTCACCTGTGATTGCTCCTAC-3′ and 5′- atagtgagtcgtattaAACGACGTTAAATTGAAAATAGGTCTA-3′. The PCR product was cloned into pJet1.2 resulting in plasmid pRB17 and sequence verified. Overlap-extension PCR was performed with 1 μl of sgRNA in vitro transcription template (see above) and 1 μl of a 10 ng/μl dilution of pRB17 per 50 μl PCR. The primers for this PCR were 5′-GCTCACCTGTGATTGCTCCTAC-3′ and 5′-gcttattctcAAAAAAGCACCGACTCGGTGCCACT-3′ (to introduce a pol-III termination signal at the end).
Affiliation: Gene Center and Dept. of Biochemistry, Ludwig-Maximilians-Universität, Feodor-Lynen-Str. 25, D-81377 München, Germany.