Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricketGryllus bimaculatus

Abstract

Studies of traditional model organisms like the fruit fly Drosophila melanogaster have contributed immensely to our understanding of the genetic basis of developmental processes. However, the generalizability of these findings cannot be confirmed without functional genetic analyses in additional organisms. Direct genome editing using targeted nucleases has the potential to transform hitherto poorly-understood organisms into viable laboratory organisms for functional genetic study. To this end, here we present a method to induce targeted genome knock-out and knock-in of desired sequences in an insect that serves as an informative contrast to Drosophila, the cricket Gryllus bimaculatus. The efficiency of germ line transmission of induced mutations is comparable to that reported for other well-studied laboratory organisms, and knock-ins targeting introns yields viable, fertile animals in which knock-in events are directly detectable by visualization of a fluorescent marker in the expression pattern of the targeted gene. Combined with the recently assembled and annotated genome of this cricket, this knock-in/knock-out method increases the viability of G. bimaculatus as a tractable system for functional genetics in a basally branching insect.

CRISPR/Cas9-Mediated genomic knock out of tyrosine hydroxylase and yellow genes in cricket Gryllus bimaculatus

“…Both of Cas9 plasmid and mRNA could not function immediately [28], they need essential time to experience a protein synthesis process in the embryo [28] and may lower the editing efficiency. In our experiments, the mutation rate is similar to that of previous Cas9 protein systems [12] and mRNA [29]. In addition, the efficiency of mutation is stable.…”

Mapping and CRISPR homology-directed repair of a recessive white eye mutation in Plodia moths

“…The impact of longer repair template insertion on knock-in efficiency is yet to be determined. Efficient insertion of larger fragments in insects have been reported using donor dsDNA template, including the use of long donor dsDNA plasmids for HDR (Gilles et al, 2015;Ma et al, 2014;Purusothaman et al, 2021), microhomology-mediated end joining in Bombyx (Nakade et al, 2014), and the homology-independent end-joining of plasmid cassettes (Bosch et al, 2020;Matsuoka et al, 2021). These approaches are promising avenues for optimizing larger cassette insertion in Plodia genome, the third lepidopteran organism after Bombyx, and Plutella where germline-heritable CRISPR HDR edits are reported (Takasu et al, 2016;Wang et al, 2020).…”

Advances in Editing Silkworms (Bombyx mori) Genome by Using the CRISPR-Cas System

“…The CRISPR-Cas9 technique was used to perform functional analysis concomitantly on two genes belonging to the cricket (Gryllus bimaculatus) [73]. G. bimaculatus is an important insect for experimental studies; for example, it plays an important role for evolutionary developmental studies and comparative biology, but it is also a relevant model organism for neurobiology and behavioral sciences [74].…”

“…After inserting the donor fragment into essential exons of both genes, their function was lost. Thus, functional investigations of hox genes could be carried out by using the knock-in/knock-out approaches [73].…”