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. 2023 Mar 24;13(4):589.
doi: 10.3390/biom13040589.

Genome and Genetic Engineering of the House Cricket (Acheta domesticus): A Resource for Sustainable Agriculture

Aaron T Dossey  1 , Brenda Oppert  2 , Fu-Chyun Chu  1 , Marcé D Lorenzen  3 , Brian Scheffler  4 , Sheron Simpson  4 , Sergey Koren  5 , J Spencer Johnston  6 , Kosuke Kataoka  7 , Keigo Ide  7
Affiliations

Genome and Genetic Engineering of the House Cricket (Acheta domesticus): A Resource for Sustainable Agriculture

Aaron T Dossey et al. Biomolecules. .

Abstract

Background: The house cricket, Acheta domesticus, is one of the most farmed insects worldwide and the foundation of an emerging industry using insects as a sustainable food source. Edible insects present a promising alternative for protein production amid a plethora of reports on climate change and biodiversity loss largely driven by agriculture. As with other crops, genetic resources are needed to improve crickets for food and other applications. Methods: We present the first high quality annotated genome assembly of A. domesticus from long read data and scaffolded to chromosome level, providing information needed for genetic manipulation. Results: Gene groups related to immunity were annotated and will be useful for improving value to insect farmers. Metagenome scaffolds in the A. domesticus assembly, including Invertebrate Iridescent Virus 6 (IIV6), were submitted as host-associated sequences. We demonstrate both CRISPR/Cas9-mediated knock-in and knock-out of A. domesticus and discuss implications for the food, pharmaceutical, and other industries. RNAi was demonstrated to disrupt the function of the vermilion eye-color gene producing a useful white-eye biomarker phenotype. Conclusions: We are utilizing these data to develop technologies for downstream commercial applications, including more nutritious and disease-resistant crickets, as well as lines producing valuable bioproducts, such as vaccines and antibiotics.

Keywords: Acheta domesticus; CRISPR-Cas9; agriculture; cricket; food security; genetic engineering; genome; insect genome; insects as food and feed; protein; sustainability.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Link density histogram of the A. domesticus Hi-C scaffolded assembly (left) and scaffold name and length of the 11 largest scaffolds in the assembly (right).
Figure 2
Figure 2
Expression profiles of three potential PGRP transcripts from A. domesticus developmental stages or male and female adults. The figure legend has the identification of transcripts from a previous transcriptome assembly [22].
Figure 3
Figure 3
Expression profiles of transcripts tentatively identified as immune-related genes in A. domesticus developmental stages or male and female adults (as indicated in the legend). Transcripts were identified in a previous transcriptome assembly [22]. (A) GNBP; (B) Lysozymes; (C) PPO.
Figure 3
Figure 3
Expression profiles of transcripts tentatively identified as immune-related genes in A. domesticus developmental stages or male and female adults (as indicated in the legend). Transcripts were identified in a previous transcriptome assembly [22]. (A) GNBP; (B) Lysozymes; (C) PPO.
Figure 3
Figure 3
Expression profiles of transcripts tentatively identified as immune-related genes in A. domesticus developmental stages or male and female adults (as indicated in the legend). Transcripts were identified in a previous transcriptome assembly [22]. (A) GNBP; (B) Lysozymes; (C) PPO.
Figure 4
Figure 4
Transposable elements (TEs) in publicly available orthopteran genomes (six crickets, including Acheta domesticus, Apteronemobius asahinai, Gryllus bimaculatus, Laupala kohalensis, Teleogryllus occipitalis, Teleogryllus oceanicus, and one locust, Locusta migratoria). (A) Contents of representative repetitive elements, including TEs, in Orthoptera. The correlation between genome coverages of major TE classes (SINE, LINE, LTR, DNA, and rolling circle) and genome sizes; (B) median length of intron; (C) Adjusted by Pearson correlation p-values (P) and coefficients (R2); (D) Repeat landscape of the major classes of TE classes (SINE, LINE, LTR, DNA, and rolling circle) in the analyzed genomes. The Kimura substitution levels (x-axis) show sequence divergence, or “TE age”, for the major classes. The classes that are skewed to the left (less sequence divergence) are estimated to have more recently diversified histories than the classes that are skewed to the right.
Figure 5
Figure 5
Design of CRISPR target in A. domesticus. (a) Schematic of the annotated Ad vermilion gene from A. domesticus genome data. Red box: ATG starting code; Yellow boxes: exons; Purple arrow: sgRNA site. Note: in this figure intron sizes are not proportional to exons; (b) Ad muscle actin gene with promoter areas. Red box: Sequences used for promoter prediction; Green sequences: predicted promoters; Red sequences: start codon; Yellow sequences: protein coding area; Gary blue area: promoter sequence used in CRISPR knock-in construct.
Figure 6
Figure 6
Vermilion eye color phenotype in A. domesticus. (A) wild-type eye color in freshly hatched cricket (left) and few hours old cricket (right). (B) Eye color of completely knocked-out Ad vermilion gene (left) and cricket with partial knock-out phenotype (right). The white arrows point out the location of the cricket eyes where the respective phenotypes can be observed.
Figure 7
Figure 7
CRISPR knock-in construct in A. domesticus. Ad muscle actin was used to identify the promoter driving EGFP as the marker gene. Two short sequences contain the Ad vermilion sgRNA sites flanking both side of the marker gene.
Figure 8
Figure 8
Somatic knock-in EGFP in A. domesticus by CRISPR/Cas9. Wild-type cricket under white light (A) and florescent light with GFP filter (B). Successful knock-in cricket with large area of somatic knock-in under white light (C) and florescent light with GFP filter (D). Cricket with small area GFP knock-in phenotype, (E) and enlarged picture of muscle structure GFP expression (F).
Figure 9
Figure 9
G1 A. domesticus with GFP expression. (A) #1 G1 eggs with some GFP expression. (B) #3 G2 eggs with GFP expression. (C) GFP crickets from #1 (left) and #3 heterozygous and homozygous (middle and right) crickets. The arrow indicates EGFP expression in the cricket leg muscle. Note: Homozygous cricket with white eye phenotype.
Figure 10
Figure 10
A. domesticus RNAi phenotypes. (A) Ad vermilion gene knock-down by RNAi in house cricket. Wild type eye color (left) compared with RNAi eye color (right). (B) GFP knock-down by RNAi in our EGFP expressing house cricket strain, AdVELV1-3. Regular EGFP expression (left) compared with dsEGFP RNAi cricket (right).
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This material is based upon work supported by the Defense Advanced Research Projects Agency (DARPA) under Contract No. 140D6318C0055. The research was completed under Cooperative Research and Development Agreement Number 58-3020-7-013 between ARS, ATB, and NCSU. This study was supported by the Cabinet Office, Government of Japan, Cross-ministerial Moonshot Agriculture, Forestry and Fisheries Research and Development Program, “Technologies for Smart Bio-industry and Agriculture” (BRAIN) [JPJ009237 to KK]. This work was supported, in part, by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health (SK). This work utilized the computational resources of the NIH HPC Biowulf cluster (https://hpc.nih.gov).
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