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CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

Xue Z, Wu M, Wen K, Ren M, Long L, Zhang X, Gao G - G3 (Bethesda) (2014)

Bottom Line: Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila.Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency.Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.

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Conditional mutation of the cid gene in the testis. (A) The phenotypes in the testis tissue are compared for the RNAi lines and CMCM lines. From top to bottom of each column: whole testis (light), tip of the testis (DAPI), seminal vesicles (DAPI), and sperm detection (DAPI). The arrows indicate testis tissue. cid:RNAi-1, BM40912; cid:RNAi-2, v43856; cid:RNAi-3, v43857; cid:RNAi-4, v109020. cid:Cas9-CM denotes testis from a conditional mutant fly via the CMCM system; nos-Gal4 was used to drive the expression of Cas9, and two vectors (U6B-cid1cid2 and CR7T-cid1cid2) were used to drive the expression of gRNA. cid:cas9-CM-1 is from U6B-cid1cid2 (28°C); cid:cas9-CM-2 is from CR7T-cid1cid2 (25°C); cid:cas9-CM-3 is from CR7T-cid1cid2 (28°C). All examined cid:Cas9-CM testes lacked mature sperm in the seminal vesicles. (B) Fertility tests of flies bearing the transgenic RNAi and the CMCM system of the cid gene. DAPI, 4′,6-diamidino-2-phenylindole.
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fig4: Conditional mutation of the cid gene in the testis. (A) The phenotypes in the testis tissue are compared for the RNAi lines and CMCM lines. From top to bottom of each column: whole testis (light), tip of the testis (DAPI), seminal vesicles (DAPI), and sperm detection (DAPI). The arrows indicate testis tissue. cid:RNAi-1, BM40912; cid:RNAi-2, v43856; cid:RNAi-3, v43857; cid:RNAi-4, v109020. cid:Cas9-CM denotes testis from a conditional mutant fly via the CMCM system; nos-Gal4 was used to drive the expression of Cas9, and two vectors (U6B-cid1cid2 and CR7T-cid1cid2) were used to drive the expression of gRNA. cid:cas9-CM-1 is from U6B-cid1cid2 (28°C); cid:cas9-CM-2 is from CR7T-cid1cid2 (25°C); cid:cas9-CM-3 is from CR7T-cid1cid2 (28°C). All examined cid:Cas9-CM testes lacked mature sperm in the seminal vesicles. (B) Fertility tests of flies bearing the transgenic RNAi and the CMCM system of the cid gene. DAPI, 4′,6-diamidino-2-phenylindole.

Mentions: We also targeted the cid (cenH3) gene, which encodes a protein essential for somatic centromere assembly and embryogenesis (Blower and Karpen 2001; Blower et al. 2006). We induced 10UAS-Cas9 expression using the nos-Gal4 driver to detect phenotypes in the testis. Testis size was greatly reduced (Figure 4A), probably because nos-Gal4 is expressed very early during spermatogenesis (Rørth 1998). Disruption of the cid gene during early spermatogenesis led to a failure in cell division and to male sterility. We could not detect any mature sperm in the seminal vesicles of the mutated testes (Figure 4A), likely explaining the observed sterility. Low temperature−induced (25°) conditional mutagenesis using both promoters resulted in a small fraction of flies with defects in testis tissue (Figure 4B). However, induced mutagenesis at 28° resulted in a high fraction of flies with defects for both promoters. Notably, all examined mutated male flies under the control of the CR7T promoter (34 of 34) were 100% sterile and showed severe phenotypes in the testes at 28° (Figure 4B).


CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

Xue Z, Wu M, Wen K, Ren M, Long L, Zhang X, Gao G - G3 (Bethesda) (2014)

Conditional mutation of the cid gene in the testis. (A) The phenotypes in the testis tissue are compared for the RNAi lines and CMCM lines. From top to bottom of each column: whole testis (light), tip of the testis (DAPI), seminal vesicles (DAPI), and sperm detection (DAPI). The arrows indicate testis tissue. cid:RNAi-1, BM40912; cid:RNAi-2, v43856; cid:RNAi-3, v43857; cid:RNAi-4, v109020. cid:Cas9-CM denotes testis from a conditional mutant fly via the CMCM system; nos-Gal4 was used to drive the expression of Cas9, and two vectors (U6B-cid1cid2 and CR7T-cid1cid2) were used to drive the expression of gRNA. cid:cas9-CM-1 is from U6B-cid1cid2 (28°C); cid:cas9-CM-2 is from CR7T-cid1cid2 (25°C); cid:cas9-CM-3 is from CR7T-cid1cid2 (28°C). All examined cid:Cas9-CM testes lacked mature sperm in the seminal vesicles. (B) Fertility tests of flies bearing the transgenic RNAi and the CMCM system of the cid gene. DAPI, 4′,6-diamidino-2-phenylindole.
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Related In: Results  -  Collection

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fig4: Conditional mutation of the cid gene in the testis. (A) The phenotypes in the testis tissue are compared for the RNAi lines and CMCM lines. From top to bottom of each column: whole testis (light), tip of the testis (DAPI), seminal vesicles (DAPI), and sperm detection (DAPI). The arrows indicate testis tissue. cid:RNAi-1, BM40912; cid:RNAi-2, v43856; cid:RNAi-3, v43857; cid:RNAi-4, v109020. cid:Cas9-CM denotes testis from a conditional mutant fly via the CMCM system; nos-Gal4 was used to drive the expression of Cas9, and two vectors (U6B-cid1cid2 and CR7T-cid1cid2) were used to drive the expression of gRNA. cid:cas9-CM-1 is from U6B-cid1cid2 (28°C); cid:cas9-CM-2 is from CR7T-cid1cid2 (25°C); cid:cas9-CM-3 is from CR7T-cid1cid2 (28°C). All examined cid:Cas9-CM testes lacked mature sperm in the seminal vesicles. (B) Fertility tests of flies bearing the transgenic RNAi and the CMCM system of the cid gene. DAPI, 4′,6-diamidino-2-phenylindole.
Mentions: We also targeted the cid (cenH3) gene, which encodes a protein essential for somatic centromere assembly and embryogenesis (Blower and Karpen 2001; Blower et al. 2006). We induced 10UAS-Cas9 expression using the nos-Gal4 driver to detect phenotypes in the testis. Testis size was greatly reduced (Figure 4A), probably because nos-Gal4 is expressed very early during spermatogenesis (Rørth 1998). Disruption of the cid gene during early spermatogenesis led to a failure in cell division and to male sterility. We could not detect any mature sperm in the seminal vesicles of the mutated testes (Figure 4A), likely explaining the observed sterility. Low temperature−induced (25°) conditional mutagenesis using both promoters resulted in a small fraction of flies with defects in testis tissue (Figure 4B). However, induced mutagenesis at 28° resulted in a high fraction of flies with defects for both promoters. Notably, all examined mutated male flies under the control of the CR7T promoter (34 of 34) were 100% sterile and showed severe phenotypes in the testes at 28° (Figure 4B).

Bottom Line: Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila.Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency.Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.

Show MeSH
Related in: MedlinePlus