Functional genetics for all: engineered nucleases, CRISPR and the gene editing revolution

Abstract

Developmental biology, as all experimental science, is empowered by technological advances. The availability of genetic tools in some species - designated as model organisms - has driven their use as major platforms for understanding development, physiology and behavior. Extending these tools to a wider range of species determines whether (and how) we can experimentally approach developmental diversity and evolution. During the last two decades, comparative developmental biology (evo-devo) was marked by the introduction of gene knockdown and deep sequencing technologies that are applicable to a wide range of species. These approaches allowed us to test the developmental role of specific genes in diverse species, to study biological processes that are not accessible in established models and, in some cases, to conduct genome-wide screens that overcome the limitations of the candidate gene approach. The recent discovery of CRISPR/Cas as a means of precise alterations into the genome promises to revolutionize developmental genetics. In this review we describe the development of gene editing tools, from zinc-finger nucleases to TALENs and CRISPR, and examine their application in gene targeting, their limitations and the opportunities they present for evo-devo. We outline their use in gene knock-out and knock-in approaches, and in manipulating gene functions by directing molecular effectors to specific sites in the genome. The ease-of-use and efficiency of CRISPR in diverse species provide an opportunity to close the technology gap that exists between established model organisms and emerging genetically-tractable species.Electronic supplementary materialThe online version of this article (doi:10.1186/2041-9139-5-43) contains supplementary material, which is available to authorized users.

Efficient CRISPR-mediated gene targeting and transgene replacement in the beetleTribolium castaneum

“…As in other experimental systems, we expect that unintended targets will occasionally be hit in the Tribolium genome (Cho et al, 2014;Fu et al, 2013;Hsu et al, 2013;Pattanayak et al, 2013). Equally, we anticipate that the technical solutions developed to reduce and control for these effects (discussed by Gilles and Averof, 2014) will also apply to Tribolium.…”

“…The bacteriophage promoters used to in vitro transcribe gRNAs also impose some additional constraints on the target sequence (reviewed by Gilles and Averof, 2014). We therefore set out to identify appropriate Tribolium promoters.…”

“…Owing to this straightforward way of generating nucleases with a chosen sequence specificity and to its high targeting efficiency in a range of organisms, CRISPR technology holds great promise for emerging model organisms (Gilles and Averof, 2014). In principle, CRISPR-mediated gene targeting should be applicable to all organisms.…”

“…Establishing efficient gene targeting in Tribolium brings to this model a more precise tool for reverse genetics, a directed approach for generating new markers and GAL4 drivers, and the opportunity to generate balancer chromosomes (for a more comprehensive discussion on the potential uses of CRISPR in emerging models, see Gilles and Averof, 2014).…”

The genome of the crustaceanParhyale hawaiensis: a model for animal development, regeneration, immunity and lignocellulose digestion

“…Crucially, genome sequence data is also necessary to further advance research in Parhyale, currently the most tractable crustacean model system. This is particularly true for the application of powerful functional genomic approaches, such as genome editing (Cong et al, 2013;Serano et al, 2015;Martin et al, 2015;Mali et al, 2013;Jinek et al, 2012;Gilles and Averof, 2014).…”