High-Efficiency CRISPR/Cas9-Mediated Gene Editing in Honeybee (Apis mellifera) Embryos

Abstract

The honeybee ( Apis mellifera ) is an important insect pollinator of wild flowers and crops, playing critical roles in the global ecosystem. Additionally, the honeybee serves as an ideal social insect model. Therefore, functional studies on honeybee genes are of great interest. However, until now, effective gene manipulation methods have not been available in honeybees. Here, we reported an improved CRISPR/Cas9 gene-editing method by microinjecting sgRNA and Cas9 protein into the region of zygote formation within 2 hr after queen oviposition, which allows one-step generation of biallelic knockout mutants in honeybee with high efficiency. We first targeted the Mrjp1 gene. Two batches of honeybee embryos were collected and injected with Mrjp1 sgRNA and Cas9 protein at the ventral cephalic side and the dorsal posterior side of the embryos, respectively. The gene-editing rate at the ventral cephalic side was 93.3%, which was much higher than that (11.8%) of the dorsal-posterior-side injection. To validate the high efficiency of our honeybee gene-editing system, we targeted another gene, Pax6 , and injected Pax6 sgRNA and Cas9 protein at the ventral cephalic side in the third batch. A 100% editing rate was obtained. Sanger sequencing of the TA clones showed that 73.3% (for Mrjp1 ) and 76.9% (for Pax6 ) of the edited current-generation embryos were biallelic knockout mutants. These results suggest that the CRISPR/Cas9 method we established permits one-step biallelic knockout of target genes in honeybee embryos, thereby demonstrating an efficient application to functional studies of honeybee genes. It also provides a useful reference to gene editing in other insects with elongated eggs.

The potential for a CRISPR gene drive to eradicate or suppress globally invasive social wasps

“…Finally, we note that should a gene drive for these wasps be desired, significant challenges still lie ahead in the production of a transgenic wasp line. The efficient production of CRISPR-Cas9 modified hymenopteran insects including honey bees ( Apis mellifera ) 56 and ants 57 offers avenues for this work.…”

CRISPR-Cas12a-Assisted Genome Editing in Amycolatopsis mediterranei

“…With CRISPR-Cas-assisted accurate cleavage in target DNA sequences and thus introducing double-stranded DNA (dsDNA) breaks (DSBs), the genome engineering efficiency can be greatly improved. Up to date, both the type II Cas9 system and type V Cas12a system have been widely applied in genome editing in a large number of species ( Cong et al, 2013 ; Jiang et al, 2014 , 2015 ; Cobb et al, 2015 ; Huang et al, 2015 ; Matsu-Ura et al, 2015 ; Low et al, 2016 ; Jia et al, 2017 ; Harrison and Hart, 2018 ; Hu et al, 2019 ). Compared to Cas9, Cas12a has several distinct features, including the preference of T-rich PAM sequences and the staggered cleavage pattern against target dsDNA ( Zetsche et al, 2015 ; Yamano et al, 2016 ).…”

Genetics in the Honey Bee: Achievements and Prospects toward the Functional Analysis of Molecular and Neural Mechanisms Underlying Social Behaviors

“…Kohno et al (2016) reported the first application of CRISPR/Cas9 in the honey bee with the production of mutant drones [63]. Currently, there are several reports of the application of CRISPR/Cas9 in the honey bee, and two procedures for analyzing gene function have been proposed [63,64,65,66].…”

“…That report was the first functional analysis using mutant honey bee workers. Xiao et al (2019) also reported highly efficient genome editing in the honey bee using similar methods [66]. Although they only analyzed embryos before they had hatched, they demonstrated that this method is effective for analyzing gene function.…”