Elucidating the composition of CRISPR-Cas12f1 complexes using mass photometry

CRISPR-Cas systems are widely used in genome engineering applications. They contain RNA-guided ribonucleoprotein (RNP) complexes that selectively modify DNA sequences at any specific location in the genome.

Due to simplified cellular delivery, miniature effector complexes such as CRISPR-Cas12f1 have become of high interest for genome engineering. However, a better understanding of the mechanisms of target recognition and DNA cleavage is essential for improving genome editing applications. Gaining this understanding is only possible if the composition of the active complex is known.

In this webinar, we describe how we addressed this question. We began by using a biophysical characterization tool, mass photometry, to measure the stoichiometry of two miniature Cas12f1 ribonucleoprotein complexes, AsCas12f1 and SpCas12f1 in vitro, as well as their interaction with nucleic acids that are involved in the genome editing process. We showed that the Cas protein forms a binary complex with guide RNA and remains bound when it interacts with target DNA – subsequently forming a ternary complex.

Overall, our results reveal the composition of a compact CRISPR-Cas system and how it affects the system’s target recognition and DNA cleavage mechanisms. These findings provide a mechanistic understanding that will be valuable for improving genome editing in the future, while also demonstrating the value of mass photometry analysis.

 

Learning Objectives