A team of researchers from the Marine Biological Laboratory, Harvard University and St. Marys College of Maryland has demonstrated efficient gene knockout in the longfin inshore squid (Doryteuthis pealeii) using CRISPR-Cas9 gene-editing system.
Crawford et al demonstrated that squid genes can be efficiently disrupted using the CRISPR-Cas9 system. Image credit: Crawford et al, doi: 10.1016/j.cub.2020.06.099.
This is a critical first step toward the ability to knock out — and knock in — genes in cephalopods to address a host of biological questions, said study senior author Dr. Joshua Rosenthal, a researcher at the Marine Biological Laboratory.
Cephalopods (squid, octopus and cuttlefish) have the largest brain of all invertebrates, a distributed nervous system capable of instantaneous camouflage and sophisticated behaviors, a unique body plan, and the ability to extensively recode their own genetic information within messenger RNA, along with other distinctive features.
These open many avenues for study and have applications in a wide range of fields, from evolution and development, to medicine, robotics, materials science, and artificial intelligence.
The ability to knock out a gene to test its function is an important step in developing cephalopods as genetically tractable organisms for biological research, augmenting the handful of species that currently dominate genetic studies, such as fruit flies, zebrafish, and mice.
It is also a necessary step toward having the capacity to knock in genes that facilitate research, such as genes that encode fluorescent proteins that can be imaged to track neural activity or other dynamic processes.
Dr. Rosenthal and colleagues used CRISPR-Cas9 genome editing to knock out a pigmentation gene in embryos of the longfin inshore squid, which eliminated pigmentation in the eye and in chromatophores (skin cells) with high efficiency.
CRISPR-Cas9 worked really well in Doryteuthis pealeii; it was surprisingly efficient, Dr. Rosenthal said.
Much more challenging was delivering the CRISPR-Cas system into the one-celled squid embryo, which is surrounded by an exceedingly tough outer layer, and then raising the embryo through hatching.
The team developed micro-scissors to clip the eggs surface and a beveled quartz needle to deliver the CRISPR-Cas9 reagents through the clip.
Ommochromes, the pigments found in squid retinas and chromatophores, are derivatives of tryptophan, and the first committed step in their synthesis is normally catalyzed by tryptophan 2,3 dioxygenase (TDO), the scientists said.
Knocking out TDO in squid embryos efficiently eliminated pigmentation.
By precisely timing CRISPR-Cas9 delivery during early development, the degree of pigmentation could be finely controlled.
The results were published in the journal Current Biology.
Karen Crawford et al. Highly Efficient Knockout of a Squid Pigmentation Gene. Current Biology, published online July 30, 2020; doi: 10.1016/j.cub.2020.06.099