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Performance of the Cas9 Nickase System in Drosophila melanogaster

Ren, Xingjie, Yang, Zhihao, Mao, Decai, Chang, Zai, Qiao, Huan-Huan, Wang, Xia, Sun, Jin, Hu, Qun, Cui, Yan, Liu, Lu-Ping, Ji, Jun-Yuan, Xu, Jiang, Ni, Jian-Quan·2014·G3 Genes|Genomes|Genetics
DOI 10.1534/g3.114.013821PMC PMC4199701
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Abstract

Recent studies of the Cas9/sgRNA system in Drosophila melanogaster genome editing have opened new opportunities to generate site-specific mutant collections in a high-throughput manner. However, off-target effects of the system are still a major concern when analyzing mutant phenotypes. Mutations converting Cas9 to a DNA nickase have great potential for reducing off-target effects in vitro. Here, we demonstrated that injection of two plasmids encoding neighboring offset sgRNAs into transgenic Cas9D10A nickase flies efficiently produces heritable indel mutants. We then determined the effective distance between the two sgRNA targets and their orientations that affected the ability of the sgRNA pairs to generate mutations when expressed in the transgenic nickase flies. Interestingly, Cas9 nickase greatly reduces the ability to generate mutants with one sgRNA, suggesting that the application of Cas9 nickase and sgRNA pairs can almost avoid off-target effects when generating indel mutants. Finally, a defined piwi mutant allele is generated with this system through homology-directed repair. However, Cas9D10A is not as effective as Cas9 in replacing the entire coding sequence of piwi with two sgRNAs.

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Figures

Figure 1 A pair of sgRNAs targeting close regions in the genome can introduce indel mutations in transgenic Cas9D10A nickase flies. (A) Diagram showing two neighboring Cas9D10A/sgRNA ribonucleoproteins targeting the fly genome. The D10A mutation converts Cas9 into a nickase that targets only the strand complementary to the sgRNA. Each Cas9D10A nickase is shown as a green circle. The sgRNA targets are shown in blue and the PAMs are shown in red. The single-strand cutting sites are shown by the magenta scissors. The offset distance is measured from the PAM-distal end of an sgRNA target to that of the other. If the PAMs are facing outward away from each other as shown in this diagram, then the distance is a positive number. (B) Scatter plot showing the relationship between heritable white mutation rate and sgRNA distance when using offset pairs in the Cas9D10A transgenic flies. Heritable mutation rate is calculated as number of the white-eyed mutant F1 flies divided by total F1 flies screened. (C) Representative sequencing results showing mutations generated with Cas9D10A transgenic flies and a pair of sgRNAs with offset distance of 11 bp. The sgRNA targets are in blue, and the PAMs are shown in red. Mutations with deleted nucleotides are shown with dashed lines, and inserted nucleotides are shown in green.
Figure 1 A pair of sgRNAs targeting close regions in the genome can introduce indel mutations in transgenic Cas9D10A nickase flies. (A) Diagram showing two neighboring Cas9D10A/sgRNA ribonucleoproteins targeting the fly genome. The D10A mutation converts Cas9 into a nickase that targets only the strand complementary to the sgRNA. Each Cas9D10A nickase is shown as a green circle. The sgRNA targets are shown in blue and the PAMs are shown in red. The single-strand cutting sites are shown by the magenta scissors. The offset distance is measured from the PAM-distal end of an sgRNA target to that of the other. If the PAMs are facing outward away from each other as shown in this diagram, then the distance is a positive number. (B) Scatter plot showing the relationship between heritable white mutation rate and sgRNA distance when using offset pairs in the Cas9D10A transgenic flies. Heritable mutation rate is calculated as number of the white-eyed mutant F1 flies divided by total F1 flies screened. (C) Representative sequencing results showing mutations generated with Cas9D10A transgenic flies and a pair of sgRNAs with offset distance of 11 bp. The sgRNA targets are in blue, and the PAMs are shown in red. Mutations with deleted nucleotides are shown with dashed lines, and inserted nucleotides are shown in green.CC BY · PubMed Central
Figure 2 Mismatched sgRNA generates heritable mutants with Cas9 nuclease, but not with Cas9D10A nickase. (A and B) Mutagenesis efficiency of sgRNAs and sgRNAs with mismatches (red). The mutagenesis efficiency of sgRNAs is tested in Cas9 or Cas9D10A transgenic flies. Each row in (A) and (B) represents an sgRNA sequence and its mutagenesis rate. (A) Mutagenesis efficiency of white-G and a white-G derivative with two mismatches. (B) Mutagenesis efficiency of white-B and a white-B derivative with one mismatch. Very low mutagenesis efficiency (<0.1%) is detected with white-G and white-B when introduced into Cas9D10A. Mismatched sgRNAs can introduce mutations when expressed in Cas9 flies, but not with Cas9D10A nickase flies.
Figure 2 Mismatched sgRNA generates heritable mutants with Cas9 nuclease, but not with Cas9D10A nickase. (A and B) Mutagenesis efficiency of sgRNAs and sgRNAs with mismatches (red). The mutagenesis efficiency of sgRNAs is tested in Cas9 or Cas9D10A transgenic flies. Each row in (A) and (B) represents an sgRNA sequence and its mutagenesis rate. (A) Mutagenesis efficiency of white-G and a white-G derivative with two mismatches. (B) Mutagenesis efficiency of white-B and a white-B derivative with one mismatch. Very low mutagenesis efficiency (<0.1%) is detected with white-G and white-B when introduced into Cas9D10A. Mismatched sgRNAs can introduce mutations when expressed in Cas9 flies, but not with Cas9D10A nickase flies.CC BY · PubMed Central
Figure 3 Mutagenesis through Cas9/sgRNA-induced HDR. (A) Diagrams showing the genomic region around the piwi locus, the donor plasmid as the repair template, and the mutation after HDR. The piwi donor piwi-4XP3-mCherry contains a 4XP3-mCherry sequence (red box) to replace most of the coding sequence of piwi. The two homologous arms (HA-L and HA-R; green) of the donor template are 0.96k bp and 1.1k bp, respectively. The piwi coding sequences are denoted by the white boxes, and the 5′ and 3′ UTRs are denoted by the shaded boxes. The Cas9/sgRNA cutting sites are denoted by the scissors. Successful replacement can be detected by mCherry expression in the fly eyes, or by PCR using the primer pairs shown as the arrows. (B) Images of w1118 control and piwiHDR-mCherry mutant flies under bright-field (top) or epifluorescent light sources (bottom). (C) Agarose gel electrophoresis result confirming successful piwi HDR mutation by PCR using primers shown in (A). (D and E) Confocal images of germaria from w1118 control (D) and piwiHDR-mCherry homozygous (piwi−/−; E) flies, stained with anti-Piwi (red), 1B1 (anti-Hts; green), and DAPI (blue). 1B1 shows the expression of Hu-li tai shao (Hts), a spectrosome/fusome protein. Note the morphology of the germarium is disrupted by extra GSC-like cells with round spectrosomes (E). Scale bars, 10 μm. Anterior, left.
Figure 3 Mutagenesis through Cas9/sgRNA-induced HDR. (A) Diagrams showing the genomic region around the piwi locus, the donor plasmid as the repair template, and the mutation after HDR. The piwi donor piwi-4XP3-mCherry contains a 4XP3-mCherry sequence (red box) to replace most of the coding sequence of piwi. The two homologous arms (HA-L and HA-R; green) of the donor template are 0.96k bp and 1.1k bp, respectively. The piwi coding sequences are denoted by the white boxes, and the 5′ and 3′ UTRs are denoted by the shaded boxes. The Cas9/sgRNA cutting sites are denoted by the scissors. Successful replacement can be detected by mCherry expression in the fly eyes, or by PCR using the primer pairs shown as the arrows. (B) Images of w1118 control and piwiHDR-mCherry mutant flies under bright-field (top) or epifluorescent light sources (bottom). (C) Agarose gel electrophoresis result confirming successful piwi HDR mutation by PCR using primers shown in (A). (D and E) Confocal images of germaria from w1118 control (D) and piwiHDR-mCherry homozygous (piwi−/−; E) flies, stained with anti-Piwi (red), 1B1 (anti-Hts; green), and DAPI (blue). 1B1 shows the expression of Hu-li tai shao (Hts), a spectrosome/fusome protein. Note the morphology of the germarium is disrupted by extra GSC-like cells with round spectrosomes (E). Scale bars, 10 μm. Anterior, left.CC BY · PubMed Central

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Citations

Cites

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Metadata

Authors
Ren, X., Yang, Z., Mao, D., Chang, Z., Qiao, H.-H., Wang, X., Sun, J., Hu, Q., Cui, Y., Liu, L.-P., Ji, J.-Y., Xu, J., & Ni, J.-Q.
Affiliations
  • Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China
  • School of Life Sciences, Tsinghua University, Beijing 100084, China
  • Tsinghua Fly Center, Tsinghua University, Beijing 100084, China
  • Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, College Station, Texas 77843
Venue
G3 Genes|Genomes|Genetics
Publisher
Oxford University Press (OUP)
Published
2014-10-01
Volume
4
Issue
10
Pages
1955-1962
ISSN
2160-1836
DOI
10.1534/g3.114.013821
PMC
PMC4199701
Cited by
40
References
7
Language
en
Licence
academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)

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