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Sci Rep. 2016 Aug 30;6:32386. doi: 10.1038/srep32386.

In vivo mutagenesis of miRNA gene families using a scalable multiplexed CRISPR/Cas9 nuclease system.

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Yale Cardiovascular Research Center, Section of Cardiology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510, USA.
Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA.
Yale Stem Cell Center and Yale Cancer Center, Yale University, New Haven, CT, 06520, USA.


A large number of microRNAs (miRNAs) are grouped into families derived from the same phylogenetic ancestors. miRNAs within a family often share the same physiological functions despite differences in their primary sequences, secondary structures, or chromosomal locations. Consequently, the generation of animal models to analyze the activity of miRNA families is extremely challenging. Using zebrafish as a model system, we successfully provide experimental evidence that a large number of miRNAs can be simultaneously mutated to abrogate the activity of an entire miRNA family. We show that injection of the Cas9 nuclease and two, four, ten, and up to twenty-four multiplexed single guide RNAs (sgRNAs) can induce mutations in 90% of the miRNA genomic sequences analyzed. We performed a survey of these 45 mutations in 10 miRNA genes, analyzing the impact of our mutagenesis strategy on the processing of each miRNA both computationally and in vivo. Our results offer an effective approach to mutate and study the activity of miRNA families and pave the way for further analysis on the function of complex miRNA families in higher multicellular organisms.

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