Gilles et al (2015) recently published a paper in Development describing CRISPR-mediated gene targeting and gene replacement in the flour beetle Tribolium castaneum.
The authors started with the Tribolium line Pig-19, which strongly expresses enhanced green fluorescent protein (EGFP) in muscle cells in larvae, pupae, and adults.
They injected Pig-19 embryos with both guide RNA targeting the EGFP coding sequence and capped Cas9 mRNA and found altered EGFP expression in 70-80% of larvae; they confirmed mutations by sequencing .
They then outcrossed the remaining G0 animals and screened progeny for altered EGFP expression, and found 71% of crosses produced non-fluorescent larvae, with loss of expression for each cross ranging from 8-100% in G1 animals.
Parents with mosaic loss of EGFP expression were more likely to transmit mutations to offspring, “suggesting that somatic targeting in G0 larvae may be a useful indicator of germline targeting in these animals.” Others, working on other insect systems seem to be making the same observation.
Similarly, targeting of the endogenous loci E-cadherin and twist was effective as verified both phenotypically and by sequencing.
The authors also were interested in using endogenous promoters to express gRNA and Cas9. They identified three U6 snRNA genes in the Tribolium genome, cloned two putative promoter fragments and placed them upstream of the eGFP1 gRNA, then injected them into Pig-19 embryos along with Cas9 mRNA. They found very similar results to injected in-vitro synthesized gRNA, suggesting endogenous promoters can be used effectively to drive in-vivo expression of gRNA and Cas9 in Tribolium.
The authors also used homology-dependent repair to generate knock-ins; specifically, they attempted to replace a green fluorescent transgene with a red transgene, with homology arms flanking the donor transgene of 0.7 kb and 1 kb (Figure 2). DsRed fluorescence was seen in 8-20% of G0 larvae, and 0-10% of second-generation larvae; they verified the conversion by sequencing.
Overall, the authors demonstrate that CRISPR / Cas9 can be used in Tribolium to make targeted knockouts and gene conversions, and that endogenous U6 promoters can be used to drive expression of guide RNAs while hsp68 effectively drove the expression of Cas9.
One drawback of these particular experiments, as the authors note, is that off-targets are not controlled for, but similar techniques to those used in Drosophila (outcrossing, avoiding off-targets in the first place), should be able to be developed relatively easily. CRISPR in Tribolium can now move forward to develop inducible and tissue-specific targeting, as well as targeting loci of interest.
This paper is worth looking at, even by those not working on Tribolium but who are thinking about using Cas9 mutagenesis and editing.
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