Various applications of TALEN- and CRISPR/Cas9-mediated homologous recombination to modify the Drosophila genome

Abstract

Modifying 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.

Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila

“…In Drosophila it has been possible for several years to create specific genome modifications by HDR, although targeting typically occurs with very low efficiency (28). Recent studies have suggested that zinc finger, transcriptional activator-like effector (TALE), and Cas9 nucleases can increase the efficiency of HDR in flies, implying that the generation of site-specific DSBs is rate limiting (11,12,27,29,30). Therefore we tested whether the highly effective creation of DSBs by our transgenic CRISPR/Cas system could be exploited to facilitate efficient HDR.…”

High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus

“…A homology-independent targeted integration (HITI) strategy without clean junctional DNA sequences has also been reported in Xenopus (Shi et al, , 2017. In all these reports the efficiency of knock-in events for small DNA sequences is low (Miyaoka et al, 2014;Yu et al, 2014), typically ∼1-2%, and although drug selection or cell sorting can be used to recover these rare integration events in cell lines, this is generally not possible in vertebrate models. As a result, CRISPR/HDR-mediated targeted insertion in animal models requires lengthy, tedious screening of F0 founders.…”

An Enhanced Gene Targeting Toolkit forDrosophila: Golic+

“…The recent introduction of CRISPR/Cas has made GT via direct injection of donor DNAs plus supporting reagents (e.g., guide RNA) into early embryos possible in Drosophila (BaenaLopez et al 2013; Gratz et al 2013;Bassett and Liu 2014;Port et al 2014;Xue et al 2014;Yu et al 2014). In addition to rapid turnover, direct embryo injection allows easy adoption of diverse GT strategies.…”

“…With CRISPR/Cas9, direct embryo injection for ends-out GT in Drosophila has been demonstrated (Baena-Lopez et al 2013;Gratz et al 2013;Port et al 2014;Xue et al 2014;Yu et al 2014) (Figure 1B). This saves the 2 months needed for the initial transgenesis of the donor DNA and may eliminate the need for complex selections.…”