In vivo mutagenesis of miRNA gene families using a scalable multiplexed CRISPR/Cas9 nuclease system
Citations Over TimeTop 10% of 2016 papers
Abstract
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.
Related Papers
- → Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos(2013)823 cited
- → CRISPR/Cas9 in zebrafish: an efficient combination for human genetic diseases modeling(2016)102 cited
- → Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio)(2018)99 cited
- → CRISPR/Cas9 disease models in zebrafish and Xenopus: The genetic renaissance of fish and frogs(2018)40 cited
- → New Developments in CRISPR/Cas-based Functional Genomics and their Implications for Research Using Zebrafish(2017)19 cited