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Somatic CRISPR-Cas9-induced mutations reveal roles of embryonically essential dynein chains in Caenorhabditis elegans cilia.

Li W, Yi P, Ou G - J. Cell Biol. (2015)

Bottom Line: Here, we report that inducible expression of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system in Caenorhabditis elegans generated conditional mutations in IFT motors and particles, recapitulating ciliary defects in their mutants.Furthermore, we demonstrate that these components undergo biphasic IFT with distinct transport frequencies and turnaround behaviors.Together, our results suggest that IFT-dynein and cytoplasmic dynein have unique compositions but also share components and regulatory mechanisms.

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Affiliation: Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.

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Conditional mutations in six embryonically essential dynein components. (A, left) The essential dynein subunits and known IFT–dynein components across species. (A, right) Gene models of dyci-1, dlc-1, dli-1, dylt-3, dyrb-1, and lis-1. Exons are in blue and red arrows indicate one or two sgRNA sequences targeting dynein components (Table S4). Bars, 300 bp. (B) Representative gels showing the results of T7EI assays. One target site generated two small fragments. (C) Embryonic lethality was determined by quantifying viable embryos after heat-shock treatment. n = 97–242; mean ± SE (error bars); *, P < 0.05; **, P < 0.01.
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fig2: Conditional mutations in six embryonically essential dynein components. (A, left) The essential dynein subunits and known IFT–dynein components across species. (A, right) Gene models of dyci-1, dlc-1, dli-1, dylt-3, dyrb-1, and lis-1. Exons are in blue and red arrows indicate one or two sgRNA sequences targeting dynein components (Table S4). Bars, 300 bp. (B) Representative gels showing the results of T7EI assays. One target site generated two small fragments. (C) Embryonic lethality was determined by quantifying viable embryos after heat-shock treatment. n = 97–242; mean ± SE (error bars); *, P < 0.05; **, P < 0.01.

Mentions: The C. elegans genome encodes 14 dynein-associated chains (Hao et al., 2011), five of which are essential for embryonic development (WormBase), including the intermediate chain (DYCI-1), light intermediate chain (DLI-1), light chain LC8 (DLC-1), Tctex-type light chain (DYLT-3), and roadblock-type light chain (DYRB-1; Fig. 2 A). We first generated transgenic animals that expressed Cas9 under the control of the Phsp promoter and ubiquitously expressed sgRNAs that target one site for dylt-3 and dyrb-1 or two sites for dyci-1, dlc-1, and dli-1 (Fig. 2 A). Our T7EI assays detected the expected molecular lesions (Fig. 2 B). Although the heat-shock treatment did not alter the embryonic viability of WT embryos, the conditional mutant embryos exhibited embryonic lethality with penetrances ranging from 40% to 55% after heat shock (Fig. 2 C). These data indicated that we generated conditional mutants of embryonically essential dynein subunits.


Somatic CRISPR-Cas9-induced mutations reveal roles of embryonically essential dynein chains in Caenorhabditis elegans cilia.

Li W, Yi P, Ou G - J. Cell Biol. (2015)

Conditional mutations in six embryonically essential dynein components. (A, left) The essential dynein subunits and known IFT–dynein components across species. (A, right) Gene models of dyci-1, dlc-1, dli-1, dylt-3, dyrb-1, and lis-1. Exons are in blue and red arrows indicate one or two sgRNA sequences targeting dynein components (Table S4). Bars, 300 bp. (B) Representative gels showing the results of T7EI assays. One target site generated two small fragments. (C) Embryonic lethality was determined by quantifying viable embryos after heat-shock treatment. n = 97–242; mean ± SE (error bars); *, P < 0.05; **, P < 0.01.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4362450&req=5

fig2: Conditional mutations in six embryonically essential dynein components. (A, left) The essential dynein subunits and known IFT–dynein components across species. (A, right) Gene models of dyci-1, dlc-1, dli-1, dylt-3, dyrb-1, and lis-1. Exons are in blue and red arrows indicate one or two sgRNA sequences targeting dynein components (Table S4). Bars, 300 bp. (B) Representative gels showing the results of T7EI assays. One target site generated two small fragments. (C) Embryonic lethality was determined by quantifying viable embryos after heat-shock treatment. n = 97–242; mean ± SE (error bars); *, P < 0.05; **, P < 0.01.
Mentions: The C. elegans genome encodes 14 dynein-associated chains (Hao et al., 2011), five of which are essential for embryonic development (WormBase), including the intermediate chain (DYCI-1), light intermediate chain (DLI-1), light chain LC8 (DLC-1), Tctex-type light chain (DYLT-3), and roadblock-type light chain (DYRB-1; Fig. 2 A). We first generated transgenic animals that expressed Cas9 under the control of the Phsp promoter and ubiquitously expressed sgRNAs that target one site for dylt-3 and dyrb-1 or two sites for dyci-1, dlc-1, and dli-1 (Fig. 2 A). Our T7EI assays detected the expected molecular lesions (Fig. 2 B). Although the heat-shock treatment did not alter the embryonic viability of WT embryos, the conditional mutant embryos exhibited embryonic lethality with penetrances ranging from 40% to 55% after heat shock (Fig. 2 C). These data indicated that we generated conditional mutants of embryonically essential dynein subunits.

Bottom Line: Here, we report that inducible expression of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system in Caenorhabditis elegans generated conditional mutations in IFT motors and particles, recapitulating ciliary defects in their mutants.Furthermore, we demonstrate that these components undergo biphasic IFT with distinct transport frequencies and turnaround behaviors.Together, our results suggest that IFT-dynein and cytoplasmic dynein have unique compositions but also share components and regulatory mechanisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.

Show MeSH
Related in: MedlinePlus