Extraordinary genetic scissors called CRISPR / Cas9, a discovery that won the Nobel Prize in Chemistry in 2020, are sometimes cut in places that are not intended for targeting. Although CRISPR has completely changed the pace of basic research by allowing scientists to quickly edit genetic sequences, it works so fast that it’s hard for scientists to see what sometimes goes wrong and figure out how to improve it.
Julene Madariaga Marcos, a Humboldt postdoctoral fellow, and colleagues in the laboratory of Professor Ralph Seidel of the University of Leipzig in Germany have found a way to analyze the ultrafast movements of CRISPR enzymes, helping researchers understand how to recognize their target sequences in hopes of improving specificity. Madariaga Marcos will present the research on Tuesday, February 23 at the 65th Annual Meeting of the Biophysical Society.
To use CRISPR enzymes to edit gene sequences, scientists can adapt them to target a specific sequence within three billion pairs of DNA bases in the human genome. During target recognition, CRISPR enzymes unwind DNA strands to find a sequence that is complementary to the CRISPR-linked RNA sequence. But sometimes RNA matches DNA sequences that don’t complement each other. To solve this unintentional match, scientists must be able to observe how CRISPR affects individual pairs of DNA bases, but the process is quick and difficult to observe.
To measure CRISPR actions in an ultra-fast time frame, Madariaga Marcos and colleagues turned to DNA origami, which uses special DNA sequences to form complex three-dimensional nanostructures instead of a simple double helix. DNA origami has applications in drug delivery, nanoelectronics, and even in the arts.
Using DNA origami, they built rotor arms from DNA to be able to watch at high speed under a microscope how the unfolding of DNA by CRISPR enzymes causes the rotor arm to rotate like helicopter blades. Using this system, they were able to measure different responses to matches and inconsistencies within a DNA sequence.
We are able to directly measure the energy landscape of a CRISPR / cascade when it first interacts with DNA. “
Julene Madariaga Marcos, postdoctoral fellow, Humboldt
This technique will help scientists better understand CRISPR enzymes and how they ultimately agree to their match. That way I can figure out how to optimize CRISPR so it makes fewer matching matches. In the future, Madariaga Marcos is interested in “developing more tools and methods to study these gene editing processes in new ways and on a more detailed level.”