Dataset Information

Non-Mendelian Dominant Maternal Effects Caused by CRISPR/Cas9 Transgenic Components in Drosophila melanogaster.

ABSTRACT: The CRISPR/Cas9 system has revolutionized genomic editing. The Cas9 endonuclease targets genomic DNA via an experimentally determined guide RNA (gRNA). This results in a double-strand break at the target site. We generated transgenic Drosophila melanogaster in which the CRISPR/Cas9 system was used to target a GAL4 transgene in vivo To our surprise, progeny whose genomes did not contain CRISPR/Cas9 components were still capable of mutating GAL4 sequences. We demonstrate this effect was caused by maternal deposition of Cas9 and gRNAs into the embryo, leading to extensive GAL4 mutations in both somatic and germline tissues. This serves as a cautionary observation on the effects of maternal contributions when conducting experiments using genomically encoded CRISPR/Cas9 components. These results also highlight a mode of artificial inheritance in which maternal contributions of DNA editing components lead to transmissible mutant defects even in animals whose genomes lack the editing components. We suggest calling this a dominant maternal effect to reflect it is caused by the gain of maternally contributed products. Models of CRISPR-mediated gene drive will need to incorporate dominant maternal effects in order to accurately predict the efficiency and dynamics of gene drive in a population.

SUBMITTER: Lin CC

PROVIDER: S-EPMC5100867 | biostudies-other | 2016 Sep

REPOSITORIES: biostudies-other

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Publications

Non-Mendelian Dominant Maternal Effects Caused by CRISPR/Cas9 Transgenic Components in Drosophila melanogaster.

Lin Chun-Chieh CC Potter Christopher J CJ

G3 (Bethesda, Md.) 20161108 11

The CRISPR/Cas9 system has revolutionized genomic editing. The Cas9 endonuclease targets DNA via an experimentally determined guide RNA (gRNA). This results in a double-strand break at the target site . We generated transgenic Drosophila melanogaster in which the CRISPR/Cas9 system was used to target a GAL4 transgene in vivo To our surprise, progeny whose genomes did not contain CRISPR/Cas9 components were still capable of mutating GAL4 sequences. We demonstrate this ...[more]

PMID: 27638686

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Project description:In nearly all metazoans, the earliest stages of development are controlled by maternally deposited mRNAs and proteins. The zygotic genome becomes transcriptionally active hours after fertilization. Transcriptional activation during this maternal-to-zygotic transition (MZT) is tightly coordinated with the degradation of maternally provided mRNAs. In Drosophila melanogaster, the transcription factor Zelda plays an essential role in widespread activation of the zygotic genome. While Zelda expression is required both maternally and zygotically, the mechanisms by which it functions to remodel the embryonic genome and prepare the embryo for development remain unclear. Using Cas9-mediated genome editing to generate targeted mutations in the endogenous zelda locus, we determined the functional relevance of protein domains conserved amongst Zelda orthologs. We showed that neither a conserved N-terminal zinc finger nor an acidic patch were required for activity. Similarly, a previously identified splice isoform of zelda is dispensable for viability. By contrast, we identified a highly conserved zinc-finger domain that is essential for the maternal, but not zygotic functions of Zelda. Animals homozygous for mutations in this domain survived to adulthood, but embryos inheriting these loss-of-function alleles from their mothers died late in embryogenesis. These mutations did not interfere with the capacity of Zelda to activate transcription in cell culture. Unexpectedly, these mutations generated a hyperactive form of the protein and enhanced Zelda-dependent gene expression. These data have defined a protein domain critical for controlling Zelda activity during the MZT, but dispensable for its roles later in development, for the first time separating the maternal and zygotic requirements for Zelda. This demonstrates that highly regulated levels of Zelda activity are required for establishing the developmental program during the MZT. We propose that tightly regulated gene expression is essential to navigate the MZT and that failure to precisely execute this developmental program leads to embryonic lethality.

Dataset Information

Non-Mendelian Dominant Maternal Effects Caused by CRISPR/Cas9 Transgenic Components in Drosophila melanogaster.

Publications

Non-Mendelian Dominant Maternal Effects Caused by CRISPR/Cas9 Transgenic Components in <i>Drosophila melanogaster</i>.

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