Abstract
ABSTRACTModifying the genomes of many organisms is becoming as easy as manipulating DNA in test tubes, which is made possible by two recently developed techniques based on either the customizable DNA binding protein, TALEN, or the CRISPR/Cas9 system. Here, we describe a series of efficient applications derived from these two technologies, in combination with various homologous donor DNA plasmids, to manipulate the Drosophila genome: (1) to precisely generate genomic deletions; (2) to make genomic replacement of a DNA fragment at single nucleotide resolution; and (3) to generate precise insertions to tag target proteins for tracing their endogenous expressions. For more convenient genomic manipulations, we established an easy-to-screen platform by knocking in a white marker through homologous recombination. Further, we provided a strategy to remove the unwanted duplications generated during the “ends-in” recombination process. Our results also indicate that TALEN and CRISPR/Cas9 had comparable efficiency in mediating genomic modifications through HDR (homology-directed repair); either TALEN or the CRISPR/Cas9 system could efficiently mediate in vivo replacement of DNA fragments of up to 5 kb in Drosophila, providing an ideal genetic tool for functional annotations of the Drosophila genome.
Highlights
In the past two years, geneticists and molecular biologists have realized the emergence of two fascinating genetic manipulation techniques, which have been shown to be applicable in essentially all animals and plants
transcription activator-like effector nuclease (TALEN)-mediated precise mutagenesis via the homology-directed repair (HDR) pathway The first application we sought to explore for TALEN and clusters of regularly interspaced short palindromic repeats (CRISPR)/Cas9 induced HDR in Drosophila was to generate precise mutagenesis in the genome
TALEN and CRISPR/Cas9 systems have been quickly adopted by biologists due to their high efficiencies in double strand break (DSB) induction and easy-to-handle procedures
Summary
In the past two years, geneticists and molecular biologists have realized the emergence of two fascinating genetic manipulation techniques, which have been shown to be applicable in essentially all animals and plants. A pair of customizable TALENs needs to be designed for a particular genomic locus that is designated for modifications (Miller et al, 2011; Zhang et al, 2011) Very recently, another method has been quickly adapted for genomic modifications in many organisms (Friedland et al, 2013; Li et al, 2013; Wang et al, 2013; Yu et al, 2013a), which is based on the Cas nuclease and a single guide RNA (gRNA) from the type II bacterial clusters of regularly interspaced short palindromic repeats (CRISPR) system (Jinek et al, 2012). Comparing with what has been reported very recently in the literature (Gratz et al, 2013), we achieved a much higher efficiency of HDR by using Lig mutant flies as recipients for injection; we directly inject DNA plasmids instead of single-strand oligonucleotides, our approach is more practical for donor preparation, especially when longer homologous sequences are needed
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