Advances in CRISPR/Cas9 Genome Editing in Drosophila Lead the Way for Applications in Other Insects.

Valentino Gantz, Ph.D. is a Faculty Assistant Research Scientist at the University of California, San Diego  His interests are in development, characterization and application of CRISPR/Cas9 gene drive technologies and related active genetic elements to benefit research and society at large. More about the author

In the January edition of FlyBook (Genetics) Bier et al. review the recent advances in CRISPR/Cas technologies applied to the fruit fly, delving into the details of established and cutting-edge methods and their potential applications to future studies in this and other insect models.

The authors cover in depth the recent literature describing the adaptation of the CRISPR/Cas genome editing system to the fruit fly and the key publications that helped to bring the technology to Drosophila. This review represents a valuable resource for researchers working with other insect organisms in the process of bringing CRISPR to their system.

Bier et al. describe well-established Drosophila CRISPR methods, from the Cas9-mediated generation of targeted mutations to the use of engineered transcription factors to modulate transcription.

Cas9 + sgRNA

Topics covered in the review include:

CRISPR/Cas components delivery: This section describes how the CRISPR components (Cas9 and guide RNA or gRNA) can be delivered (transgenic, plasmid or capped mRNA) and the advantages and disadvantages of each method.

Targeted mutagenesis and deletions: The use of engineered gRNA/Cas9 can be used to target a double strand DNA cut to any region of interest in the genome. The endogenous error-prone repair pathway “Non-Homologous End-Joining” or NHEJ can generate small insertion/deletions at the target location. Additionally, when two nearby gRNAs are used, deletions of the intervening sequence can be efficiently recovered. This method has been used to interrogate the function of genes, both in the coding region and its regulatory sequences.

DNA repair following double-stranded breaks

Insertions and replacements: By co-opting a second, highly accurate, endogenous DNA-repair pathway, “Homology-Directed Repair” or HDR it is possible to generate precise insertions and deletion in the fruit fly genome, allowing for unprecedented flexibility in genome editing. These methods use exogenous templates carrying homology to the region of interest to repair the Cas9-generated break. While these strategies require the addition of a third variable (DNA repair template), they exploit the HDR pathway which is well-conserved from yeast to human as well as every insect in between.

CRISPRi and CRISPRa: These applications use engineered transcription factors built with a nuclease-dead form of the Cas9 enzyme, still capable of binding the DNA in a gRNA-dependent fashion, and a transcription inhibitor or activator respectively. gRNAs can be designed to localize these proteins in the vicinity of promoter sequences allowing researchers to modulate at will the transcription of genes of interest.

A selfish gene (blue) spreading through a mosquito population

Active Genetics and Gene Drive: Optimized CRISPR tools in Drosophila have been used to develop a novel technology based on autocatalytic genetic elements capable of cutting the and copying themselves onto the opposing chromosome at the same exact genomic location. The copying process, driven by the presence of a germline source of Cas9, dramatically increases the frequency of inheritance in the progeny of the element of interest, permitting a fast assembly of multiple transgenes in the same individual for research purposes. Additionally, when both the gRNA and the Cas9 genes are inserted at the same location that the gRNA is targeting, the genetic element can be used to generate a strong gene drive effect. This property can be utilized for population modification or suppression strategies to combat vector-borne diseases carried by mosquitoes or other insects, crop pests, or invasive species.

Bier, E., Harrison, M. H., O’Connor-Giles, K. M., and J. Wildonger (2018). Advances in Engineering the Fly Genome with the CRISPR-Cas System. Genetics 208:1-18;

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