Awata et al. (2015) have used Cas9 to knock-out Dop1 in Gryllus bimaculatus to assess the role of dopamine neurons in mediating reward and punishment.
Functional genomics technologies like transposons and first and second generation gene-editing technologies have been available for use in Gryllus bimaculatus and, as with other insects, require microinjection of early embryos in order to deliver DNA, RNA and proteins to cells giving rise to germ cells.
Here Awata and colleagues use Cas9 endonuclease from the Streptococcus pyogenes CRISPR system to created a null allele of the dopamine receptor gene, Dop1.
Awata et al. chose a 20 bp target region followed by an NGG PAM in the second exon of Dop1. They used ZiFiT Targeter Version 4.2 to choose a target. ZiFiT was originally designed to aid in the creation of zinc finger nucleases and has be updated as other editing technologies have become available and now can be used to TALEN and CRISPR studies.
Guide RNAs were constructed using standard PCR based methods in conjunction with in vitro transcription. Cas9 mRNA was also produced by in vitro transcription, capping and polyadenylation. Guide RNA and Cas9 mRNA were injected into embryos ≤ 5hrs after oviposition with each at 500ng/ul.The authors injected 15 embryos from which 5 survived to adults. These were mated with wild-type individuals and the F1 were screened for the presence of Dop1 mutations by isolating genomic DNA from the foreleg of the tibia and performing a PCR that allowed for the detection indels.
One strain produced mutant progeny and the line established had a complex mutation consisting of a small deletion in the target sequence as well as small deletions and insertions and two ~100 bp duplicated insertions near the target region. It is not clear how this complex mutation may have arisen but it resulted in knocking out transcription of the gene and not simply a disfunctional mRNA. A homozygous line was created using foreleg tibia genotyping as a way to identify individuals of interest.
Off-target effects are a concern for these types of experiments and in this case Awata et al. searched the
genome for sequences that were similar to the guide RNA and checked these loci by PCR and sequencing in the homozygous mutant line and found that those loci were wild-type.
So, using delivery and screening strategies developed earlier, the authors were able to create a knock-out line with a relatively small up front effort to deliver Cas9 to 15 embryos. That’s pretty encouraging, if you work on Gryllus.
While the technical aspects of the paper are interesting, the analysis of Dop1 homozygotes is the paper’s raison d’être and you should consult the paper for those interesting finding.
Hiroko Awata, Takahito Watanabe, Yoshitaka Hamanaka, Taro Mito, Sumihare Noji & Makoto Mizunami (2015) Knockout crickets for the study of learning and memory: Dopamine receptor Dop1 mediates aversive but not appetitive reinforcement in crickets. Scientific Reports 5, Article number: 15885 (2015) doi:10.1038/srep15885