Efficient CRISPR/Cas9 Knockout of vermillion in Helicoverpa zea (Boddie)


Thais Souza, Ph.D. is a Postdoctoral Researcher at the University of Sao Paulo and the University of California Riverside where she doing research to understand how polyphagous insects can overcome the effects of plant peptidase inhibitors (PPI). She been studying the molecular interactions between PPIs and serine peptidase genes present in insect midguts and is developing gene editing tools to investigate these questions in Spodoptera frugiperda. More about the Author

Perera et al. (2018) recently used CRISPR/Cas gene editing in Helicoverpa zea, a lepidopteran pest of many crops. The authors showed positive correlations between different concentrations of ribonucleoprotein (RNP) complexes and Cas9 protein with mutation percentage of the vermillion gene in H. zea.

Insect pests cause extensive agricultural losses worldwide. Because of incorrect use of chemical insecticides and inappropriate practices of integrated pest management (IPM), insects are developing high resistance to insecticides and other forms of control (e.g., transgenic plants). Molecular genetic technologies can contribute to improved management of insects.

File:Helicoverpa zea larva.jpg

Heliothis (Helicoverpa) zealarvae on corn. Image licensed under the terms of the cc-by-sa-2.0

Genome editing with the CRISPR/Cas9 system is a potential alternative resource for species-specific control. New research on optimization of gene editing, including verification of the CRISPR/Cas9 efficiency to induce targeted mutagenesis, are crucial for this technique’s applicability in non-model insects.

The eye color in insects is produced by two classes of pigments; pteridines and ommochromes. The vermillion gene is responsible for encoding the first enzyme in the ommochrome synthesis pathway, the tryptophan 2,3-dioxygenase (TO). The lack of TO enzyme in the ommochrome pathway causes a reduction in brown pigments (ommochromes) and leaving mostly red pteridines, resulting in bright red or yellow eyes.

eye pigments

Ommochrome and pteridine biosynthetic pathways with notable mutations shown. Kamleh et al., Towards a platform for the metabonomic profiling of different strains of Drosophila melanogaster …, The FEBS Journal, (2009) doi:10.1111/j.1742-4658.2009.07397.x Image by David Swift.

Perera et al designed two sgRNAs based on the sequence of the TO gene sequence in H. zea.  Ribonucleoprotein(RNP) complexes consisting of sgRNAs and Cas9 protein were injected into H. zea eggs early in development.  Three different sgRNA concentrations were used (1μM, 2μM and 4μM) and the results showed that RNPs assembled with  1μM sgRNA concentrations produced fewer mutants than RNPs assembled with 2μM or 4μM sgRNA concentrations and no deleterious effects were found on embryo viability. H. zea TO mutants have yellow eyes color or pink eye color that gradually changed to yellow. Compared with other studies in non-model insects that employed transcribed gRNA with Cas protein, the mutation rates observed by Perera et al. (~90%) were quite high.

The main difference in this study compared to other insect gene editing studies was the use of Cas9 nuclease containing nuclear localization signals (NLS).  The inclusion of nuclear localization signals has been shown to improve editing efficiency in non-insect systems.

Nuclear localization signals have been added to Cas9 in a variety of positions. More about Cas9-NLS

The nucleotide sequences showed different combinations of deletions and insertions in mutant phenotypes. Interestingly, all the insects that were classified as having wild type eye color in G0 showed some type of mutation in one or both target sites. Potential off-targets were identified in H. zea genome. However, PCR amplification and nucleotide sequencing did not show any unintended mutations.

In addition, the authors highlight the challenges in comparing studies in non-model insects and suggest guidelines to promote a standardization experiments of CRISPR/Cas mediated genome editing.


Perera OP, Little NS, Pierce CA, III (2018) CRISPR/Cas9 mediated high efficiency knockout of the eye color gene Vermillion in Helicoverpa zea (Boddie). PLoS ONE 13(5): e0197567. https://doi.org/10.1371/journal.pone.0197567


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