Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-Associated Protein and Its Utility All at Sea: Status, Challenges, and Prospects

Abstract

Initially discovered over 35 years ago in the bacterium Escherichia coli as a defense system against invasion of viral (or other exogenous) DNA into the genome, CRISPR/Cas has ushered in a new era of functional genetics and served as a versatile genetic tool in all branches of life science. CRISPR/Cas has revolutionized the methodology of gene knockout with simplicity and rapidity, but it is also powerful for gene knock-in and gene modification. In the field of marine biology and ecology, this tool has been instrumental in the functional characterization of 'dark' genes and the documentation of the functional differentiation of gene paralogs. Powerful as it is, challenges exist that have hindered the advances in functional genetics in some important lineages. This review examines the status of applications of CRISPR/Cas in marine research and assesses the prospect of quickly expanding the deployment of this powerful tool to address the myriad fundamental marine biology and biological oceanography questions.

Keywords: CRISPR/Cas; functional genetic research; genome editing technology; marine biology; marine prokaryotic microbes; phytoplankton; zooplankton.

Figure 1

Overview of the CRISPR–Cas immune system. Spacer acquisition: the spacer sequence from the virus is sampled and then integrated into the CRISPR locus. Expression: Pre-crRNA is transcribed from the leader region and processed into smaller crRNAs by Cas proteins. Target degradation: the crRNA and Cas endonuclease complex identifies invading nucleic acid (viral or plasmid) sequences and initiates a cleavage event.

Figure 2

Workflow of CRISPR/Cas-based genome editing.

Figure 3

Maximum likelihood tree of the Cas9, Cas12, and Fanzor proteins from bacteria, fungus, and dinoflagellate. Color shading depicts cluster of Cas subtype named on the right. Bootstrap values on the trees were derived from 1000 resampling. In red font are eukaryotes, corresponding to Fanzor from fungi and Cas9 from dinoflagellates.

Figure 4

Roadmap for future applications of CRISPR/Cas in marine biological research.