One of the most powerful tools available to biologists these days is CRISPR-Cas9, a combination of specialized RNA and protein that acts like a molecular scalpel, allowing researchers to precisely slice and dice pieces of an organism's genetic code.
But even though CRISPR-Cas9 technology has offered an unprecedented level of control for those studying genetics and genetic engineering, there has been room for improvement. Now, a new technique developed at Caltech by biology graduate student Shashank "Sha" Gandhi in the lab of Marianne Bronner, Distinguished Professor of Biology and director of the Beckman Institute, is taking CRISPR-Cas9 accessibility to the next level.
In a paper appearing in the journal Development, Gandhi and members of Bronner's lab describe the new technique, which has been designed specifically to disable or remove genes from a genome. This is known as "knocking out" a gene.
Gene knockout is an important method for studying what genes do because researchers can compare the behavior of a cell that has a working gene to the behavior of a cell in which that gene has been disabled. CRISPR-Cas9 has already been used for this, usually alongside genetic material that encodes a fluorescent protein, which makes it easy to identify cells from which a gene has been removed; cells with a knocked-out gene will glow.
One drawback of the technique, however, is that each part of the payload that makes it work—the Cas9 protein, the guide RNA, and the code for the fluorescent protein—have to be delivered separately using a technique called electroporation, which opens the membranes of cells by zapping them with electricity. This can result in some cells receiving only some of the pieces. Thus, a cell could receive the code for making fluorescent protein, but not end up with a knocked-out gene. Or a cell could end up with its targeted gene knocked out, but not have the code to make fluorescent protein. Either case makes it more challenging for researchers to study how the cells are behaving.
