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Performance of the Cas9 nickase system in Drosophila melanogaster.

Ren X, Yang Z, Mao D, Chang Z, Qiao HH, Wang X, Sun J, Hu Q, Cui Y, Liu LP, Ji JY, Xu J, Ni JQ - G3 (Bethesda) (2014)

Bottom Line: Mutations converting Cas9 to a DNA nickase have great potential for reducing off-target effects in vitro.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.However, Cas9(D10A) is not as effective as Cas9 in replacing the entire coding sequence of piwi with two sgRNAs.

View Article: PubMed Central - PubMed

Affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.

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Related in: MedlinePlus

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.
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fig3: 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.

Mentions: Because Cas9D10A can efficiently generate indel mutants through the NHEJ pathway, we then evaluated the efficiency of Cas9D10A in HDR. Wild-type Cas9 nuclease had been successfully applied in generating mutants through HDR (Baena-Lopez et al. 2013; Gratz et al. 2014; Yu et al. 2014; Xue et al. 2014). Ideally, complete loss-of-function mutants need to dispose of entire coding sequences. To precisely replace the entire coding sequence, the approach we developed involved the transgenic Cas9 flies, two sgRNAs, and a plasmid DNA donor with a selective marker (Materials and Methods; Figure 3A).


Performance of the Cas9 nickase system in Drosophila melanogaster.

Ren X, Yang Z, Mao D, Chang Z, Qiao HH, Wang X, Sun J, Hu Q, Cui Y, Liu LP, Ji JY, Xu J, Ni JQ - G3 (Bethesda) (2014)

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.
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Related In: Results  -  Collection

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fig3: 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.
Mentions: Because Cas9D10A can efficiently generate indel mutants through the NHEJ pathway, we then evaluated the efficiency of Cas9D10A in HDR. Wild-type Cas9 nuclease had been successfully applied in generating mutants through HDR (Baena-Lopez et al. 2013; Gratz et al. 2014; Yu et al. 2014; Xue et al. 2014). Ideally, complete loss-of-function mutants need to dispose of entire coding sequences. To precisely replace the entire coding sequence, the approach we developed involved the transgenic Cas9 flies, two sgRNAs, and a plasmid DNA donor with a selective marker (Materials and Methods; Figure 3A).

Bottom Line: Mutations converting Cas9 to a DNA nickase have great potential for reducing off-target effects in vitro.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.However, Cas9(D10A) is not as effective as Cas9 in replacing the entire coding sequence of piwi with two sgRNAs.

View Article: PubMed Central - PubMed

Affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.

Show MeSH
Related in: MedlinePlus