A groundbreaking method for introducing tiny research instruments into egg cells and embryos has emerged, overcoming a significant hurdle in applying the CRISPR-Cas9 gene-editing technology to a variety of research organisms.
Researchers at the Marine Biological Laboratory (MBL) have devised a novel approach known as VitelloTag for delivering essential tools into egg cells and embryos, effectively addressing a critical challenge in utilizing the CRISPR-Cas9 gene-editing system across numerous research organisms. This innovative method has been detailed in a paper accepted for publication by the journal Development.
Typically, scientists rely on long, slender glass needles to inject their research materials, a process called microinjection which demands considerable time and skill. Additionally, this method sometimes faces obstacles due to the nature of the cells involved; for example, cephalopod egg cells are shielded by a tough outer layer known as chorion, which causes glass needles to shatter upon contact.
“For some organisms, you need to craft ultra-sharp gourmet needles for effective microinjection, and they can still break,” shared Zak Swartz, an assistant scientist at MBL and the lead author of the VitelloTag publication.
Other species present further challenges, with delicate embryos or exceedingly brief spawning periods limiting the opportunities for successful CRISPR-Cas9 delivery into their eggs. Consequently, the success rate using microinjection is often quite low.
The new technique was inspired by vitellogenin, a yolk protein prevalent in most animals that serves as an energy reserve for developing eggs.
“The fascinating biological aspect, which underlies this tool, is that vitellogenin is produced outside the ovary,” explained Swartz. “In chickens, for example, it originates in the liver and is then transported through the bloodstream to reach the ovary, where it enters the egg cell. Our goal was to harness this biological process to create a method for delivering materials into egg cells.”
While vitellogenin is a large protein, the research team was able to isolate its smaller segment that attaches to receptors on the egg cell surface. “This small tag—comprising about 10 amino acids—can be coupled with various materials like Cas9,” Swartz stated. “This means you can have your eggs in a petri dish, introduce the VitelloTag linked to Cas9, and the eggs will absorb it in bulk, eliminating the need for tedious individual microinjections.”
The team has effectively utilized VitelloTag in two diverse species critical for developmental biology: the sea star (Patiria miniata) and the acorn worm (Saccoglossus kowalevskii). Since the paper was made available online, “we’ve seen a surge of interest from various institutions wanting to try it on their specific organisms,” Swartz added.
Vitellogenin is highly preserved across different animal species, indicating that the tool could potentially function well across a broad range of organisms. “However, we have developed a process to create customized versions of VitelloTag tailored to specific species if the initial version does not perform as intended,” Swartz remarked.
Despite this innovation, microinjection remains the preferred method for delivering CRISPR-Cas9 in many organisms, achieving a penetrance (the percentage of cells that successfully absorb the CRISPR material) as high as 90 percent. In contrast, the team obtained around 30 percent penetrance using VitelloTag with the sea star and acorn worm.
“Even if you can rapidly apply VitelloTag to a dish of 500 eggs and achieve 30 percent penetrance, it’s still quite effective,” said Swartz. “For animals like the acorn worm, where injection proves particularly challenging, VitelloTag represents a significant improvement. It offers far better success rates than were previously possible.”
The research collaborates with D. Nathaniel Clarke from M.I.T, along with Akshay Kane, Margherita Perillo from the MBL, and Christopher J. Lowe from the Hopkins Marine Station at Stanford University.
