The CRISPR/Cas9 technique is widely used in experimentation with human cell lines as well as with other model systems, such as mice Mus musculus, zebrafish Danio reiro, and the fruit fly Drosophila melanogaster. However, publications describing the use of CRISPR/Cas9 for genome editing in non-model organisms, including non-model insects, are scarce. The introduction of this relatively new method presents many problems even for experienced researchers, especially with the lack of procedures to tackle issues concerning the efficiency of mutant generation. Here we present a protocol for efficient genome editing in the non-model insect species Pyrrhocoris apterus. We collected data from several independent trials that targeted several genes using the CRISPR/Cas9 system and determined that several crucial optimization steps led to a remarkably increased efficiency of mutant production. The main steps are as follows: the timing of embryo injection, the use of the heteroduplex mobility assay as a screening method, in vivo testing of sgRNA efficiency, and G0 germline mosaicism screening. The timing and the method of egg injections used here need to be optimized for other species, but other here-described optimization solutions can be applied immediately for genome editing in other insect species.

Department of Molecular Biology Faculty of Sciences University of South Bohemia České Budějovice Czechia

Laboratory of Molecular Chronobiology Department of Molecular Biology and Genetics Institute of Entomology Biology Centre Czech Academy of Sciences České Budějovice Czechia

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Impact of photoperiod and functional clock on male diapause in cryptochrome and pdf mutants in the linden bug Pyrrhocoris apterus

Evolution of casein kinase 1 and functional analysis of new doubletime mutants in Drosophila

Pigment Dispersing Factor Is a Circadian Clock Output and Regulates Photoperiodic Response in the Linden Bug, Pyrrhocoris apterus

Loss of Timeless Underlies an Evolutionary Transition within the Circadian Clock

Functional analysis and localisation of a thyrotropin-releasing hormone-type neuropeptide (EFLa) in hemipteran insects

Light and Temperature Synchronizes Locomotor Activity in the Linden Bug, Pyrrhocoris apterus

New Drosophila Circadian Clock Mutants Affecting Temperature Compensation Induced by Targeted Mutagenesis of Timeless