Using CRISPR/Cas9 to investigate the role of candidate human disease gene orthologs in Ciona

Functional Genomics in Zoology: Harnessing CRISPR-Cas Mechanisms and Applications

The advent of CRISPR–Cas genome editing has changed the study of animal biology, turning functional genomics from a discipline constrained by slow forward-genetic screens and bespoke, species-specific reagents into one capable of rapid, programmable, and broadly portable genetic manipulation. This review critically synthesises the past decade of progress in applying CRISPR-based tools across zoology, spanning classical vertebrate model organisms, emerging non-model invertebrates, livestock and aquaculture species, and wildlife populations of direct conservation concern. We examine the expanding molecular toolkit, from nuclease-mediated knockouts through base and prime editing to transcriptional and epigenetic modulation, and consider how these tools have been adapted to taxa as different as zebrafish, cephalopods, reef-building corals, lepidopteran insects, poultry, and large-bodied mammals. Particular attention is given to three areas where functional genomics meets applied zoology and conservation biology most directly: genetic rescue and de-extinction science aimed at restoring genetic diversity or ecological function to imperilled or vanished species; gene drive technologies designed to suppress or modify wild populations of disease vectors and invasive pests; and genome-edited livestock, poultry, and aquaculture stocks engineered for disease resistance, welfare improvement, and production efficiency. We further evaluate methodological advances in detecting and mitigating off-target mutagenesis, the welfare and biosafety implications of intentionally altering the genomes of sentient animals, and the evolving regulatory landscape governing genome-edited organisms in agriculture, biomedicine, and the environment. Throughout, we take a critical stance, weighing demonstrated efficacy against persistent technical limitations, ecological uncertainty, and unresolved ethical questions. We conclude that CRISPR-based functional genomics has reshaped zoological enquiry in ways unlikely to be reversed, yet its translation into field-deployed conservation and agricultural interventions remains constrained by incomplete ecological risk assessment, uneven regulatory harmonisation, and the biological idiosyncrasies of non-model taxa that resist easy extrapolation from laboratory systems.

Compact type II-D Cas9 nucleases for efficient and specific genome editing

Compact CRISPR nucleases are attractive for therapeutic genome editing because their small coding sequences facilitate delivery by adeno-associated virus. Type II-D Cas9 (Cas9d) enzymes constitute the most compact Cas9 subtype, yet only a few orthologs have demonstrated mammalian genome-editing activity, leaving it unclear whether this activity is general or exceptional. Here, we mined the IMG/M metagenomic database and identified five previously uncharacterized MG102-like Cas9d orthologs (∼950 amino acids) that share the hallmark genomic, sequence, and structural features of type II-D Cas9. Two of them, Cas9d-1 and Cas9d-4, recognized a 5’-NRC-3’ protospacer-adjacent motif and edited endogenous human loci with efficiencies up to 20.1%, exceeding Streptococcus pyogenes Cas9 at one site, while producing deletion-biased outcomes and no detectable off-target activity. Notably, both orthologs edited more efficiently than the sole previously validated member of this lineage, MG102-2, when assayed side by side under identical conditions. These findings establish compact MG102-like Cas9d orthologs as robust and specific genome editors and provide promising, single-AAV– compatible scaffolds for in vivo therapeutic genome editing.

Ef fi cient epigenetic editing of the zebra fi sh slc45a2 gene using the CRISPR/dCas9 system and tandem guide RNAs

Establishment of a CRISPR/Cas9-mediated system for targeted editing of the MFS gene in mint

A substantial decrease in menthofuran content in the essential oil of the edited line #10 compared to the wild-type control is revealed, thereby demonstrating a viable strategy for improving mint essential oil quality through genome-editing.

CRISPR/Cas9-Mediated Disruption of Duplicated Sizzled Genes Induces Twin-Tail-like Caudal Bifurcation in Goldfish (Carassius auratus)

Findings provide direct functional evidence that szl regulates median caudal patterning in goldfish and suggest that szl-dependent modulation of the Chordin/BMP network can generate twin-tail-like caudal morphology.

CRISPR/Cas9-Based Genome Editing: Understanding Differences in DNA Repair Pathways, Profiles, and Outcomes

Over a decade of advances in Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-based technologies have culminated in the first-ever FDA-approved CRISPR/Cas-based therapy. Aside from this approved therapy for sickle cell anemia, several CRISPR/Cas-based therapies are currently under development or testing for a range of chronic diseases, including viral diseases like human immunodeficiency virus type 1 (HIV-1) infection, genetic diseases like familial hypercholesterolemia, and cancer. The success of these therapies hinges on the effective delivery of CRISPR/Cas9 components to target regions, efficient Cas endonuclease editing, repair profiles generated, and their resulting outcomes. Here, we discuss the factors that influence the generation of CRISPR/Cas9-generated repair edits, the overall profiles, and outcome prediction(s), as well as the analytical tools that have been developed to date. Finally, how this technology has been used towards a functional HIV-1 cure is discussed.