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Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells.

Böttcher R, Hollmann M, Merk K, Nitschko V, Obermaier C, Philippou-Massier J, Wieland I, Gaul U, Förstemann K - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Gene Center and Dept. of Biochemistry, Ludwig-Maximilians-Universität, Feodor-Lynen-Str. 25, D-81377 München, Germany.

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Quantitative assessment of cas9-mediated cleavage in living cells using various CRISPR RNA guides. (A) Sequence detail of the I-SceI recognition site region in our reporter construct. The length variant CRISPR RNAs with an NGG trinucleotide protospacer-associated motif (PAM) are shown in blue below the reporter sequence and the CRISPR RNA for testing cleavage efficiency at the 5'-NAG-3' trinucleotide PAM is shown in red above the reporter sequence. (B) Quantitative analysis of CRISPR cleavage activity according to targeting sequence length using our HR reporter system. The proportion of GFPhigh cells was determined via two-dimensional analysis of fluorescence-activated cell sorting data (side scatter vs. GFP fluorescence) as this enables more reliable separation of the two populations. Two independent cell clones expressing myc-cas9 and the double-GFP reporter were analyzed; the data are presented as the mean ±SD of three independent biological replicates. (C) Quantitative analysis of CRISPR cleavage activity with an NAG trinucleotide PAM. The experiment was performed essentially as described in B, the I-Sce17 NGG CRISPR serves as comparison. Cleavage activity towards an NAG PAM is detectable but occurs with clearly lower efficiency. Two cell clones were tested in two independent biological replicates each. (D) Extending the CRISPR RNA at its 3′-end does not impair cleavage activity. The experiment was performed and analyzed as in B but a CRISPR RNA with a 3′-extension harboring 18 nt of sequence homology downstream of the CRISPR target site was employed. This extension did not impair cleavage activity (compare with B), but also did not rescue the apparent defect of the I-Sce13GG RNA construct either.
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Figure 2: Quantitative assessment of cas9-mediated cleavage in living cells using various CRISPR RNA guides. (A) Sequence detail of the I-SceI recognition site region in our reporter construct. The length variant CRISPR RNAs with an NGG trinucleotide protospacer-associated motif (PAM) are shown in blue below the reporter sequence and the CRISPR RNA for testing cleavage efficiency at the 5'-NAG-3' trinucleotide PAM is shown in red above the reporter sequence. (B) Quantitative analysis of CRISPR cleavage activity according to targeting sequence length using our HR reporter system. The proportion of GFPhigh cells was determined via two-dimensional analysis of fluorescence-activated cell sorting data (side scatter vs. GFP fluorescence) as this enables more reliable separation of the two populations. Two independent cell clones expressing myc-cas9 and the double-GFP reporter were analyzed; the data are presented as the mean ±SD of three independent biological replicates. (C) Quantitative analysis of CRISPR cleavage activity with an NAG trinucleotide PAM. The experiment was performed essentially as described in B, the I-Sce17 NGG CRISPR serves as comparison. Cleavage activity towards an NAG PAM is detectable but occurs with clearly lower efficiency. Two cell clones were tested in two independent biological replicates each. (D) Extending the CRISPR RNA at its 3′-end does not impair cleavage activity. The experiment was performed and analyzed as in B but a CRISPR RNA with a 3′-extension harboring 18 nt of sequence homology downstream of the CRISPR target site was employed. This extension did not impair cleavage activity (compare with B), but also did not rescue the apparent defect of the I-Sce13GG RNA construct either.

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).


Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells.

Böttcher R, Hollmann M, Merk K, Nitschko V, Obermaier C, Philippou-Massier J, Wieland I, Gaul U, Förstemann K - Nucleic Acids Res. (2014)

Quantitative assessment of cas9-mediated cleavage in living cells using various CRISPR RNA guides. (A) Sequence detail of the I-SceI recognition site region in our reporter construct. The length variant CRISPR RNAs with an NGG trinucleotide protospacer-associated motif (PAM) are shown in blue below the reporter sequence and the CRISPR RNA for testing cleavage efficiency at the 5'-NAG-3' trinucleotide PAM is shown in red above the reporter sequence. (B) Quantitative analysis of CRISPR cleavage activity according to targeting sequence length using our HR reporter system. The proportion of GFPhigh cells was determined via two-dimensional analysis of fluorescence-activated cell sorting data (side scatter vs. GFP fluorescence) as this enables more reliable separation of the two populations. Two independent cell clones expressing myc-cas9 and the double-GFP reporter were analyzed; the data are presented as the mean ±SD of three independent biological replicates. (C) Quantitative analysis of CRISPR cleavage activity with an NAG trinucleotide PAM. The experiment was performed essentially as described in B, the I-Sce17 NGG CRISPR serves as comparison. Cleavage activity towards an NAG PAM is detectable but occurs with clearly lower efficiency. Two cell clones were tested in two independent biological replicates each. (D) Extending the CRISPR RNA at its 3′-end does not impair cleavage activity. The experiment was performed and analyzed as in B but a CRISPR RNA with a 3′-extension harboring 18 nt of sequence homology downstream of the CRISPR target site was employed. This extension did not impair cleavage activity (compare with B), but also did not rescue the apparent defect of the I-Sce13GG RNA construct either.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4066747&req=5

Figure 2: Quantitative assessment of cas9-mediated cleavage in living cells using various CRISPR RNA guides. (A) Sequence detail of the I-SceI recognition site region in our reporter construct. The length variant CRISPR RNAs with an NGG trinucleotide protospacer-associated motif (PAM) are shown in blue below the reporter sequence and the CRISPR RNA for testing cleavage efficiency at the 5'-NAG-3' trinucleotide PAM is shown in red above the reporter sequence. (B) Quantitative analysis of CRISPR cleavage activity according to targeting sequence length using our HR reporter system. The proportion of GFPhigh cells was determined via two-dimensional analysis of fluorescence-activated cell sorting data (side scatter vs. GFP fluorescence) as this enables more reliable separation of the two populations. Two independent cell clones expressing myc-cas9 and the double-GFP reporter were analyzed; the data are presented as the mean ±SD of three independent biological replicates. (C) Quantitative analysis of CRISPR cleavage activity with an NAG trinucleotide PAM. The experiment was performed essentially as described in B, the I-Sce17 NGG CRISPR serves as comparison. Cleavage activity towards an NAG PAM is detectable but occurs with clearly lower efficiency. Two cell clones were tested in two independent biological replicates each. (D) Extending the CRISPR RNA at its 3′-end does not impair cleavage activity. The experiment was performed and analyzed as in B but a CRISPR RNA with a 3′-extension harboring 18 nt of sequence homology downstream of the CRISPR target site was employed. This extension did not impair cleavage activity (compare with B), but also did not rescue the apparent defect of the I-Sce13GG RNA construct either.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Gene Center and Dept. of Biochemistry, Ludwig-Maximilians-Universität, Feodor-Lynen-Str. 25, D-81377 München, Germany.

Show MeSH
Related in: MedlinePlus