J Kornherr(1), N Holzleitner(1), C Plumeyer(1), S Ottonello(1), S Koscielniak(1), C Poch(1), C Kupatt-Jeremias(1), KL Laugwitz(1), A Moretti(1), J Grünewald(1)

1: Department of Cardiology, TUM University Hospital - Klinikum rechts der Isar, Technical University of Munich / DZHK (German Center for Cardiovascular Research)

CRISPR-guided Cas9 and Cas12 proteins enable RNA-targeted DNA binding and/or cleavage, as well as several “CRISPR 2.0” applications, such as epigenetic, base, and prime editing. However, the most commonly used Cas proteins are relatively large in size, complicating in vivo delivery. Here, we describe a combination of structure-guided and AI-based protein engineering strategies to increase the gene editing functionalities of a miniature type V system in human cells. Based on this, we aim to develop a simple to use and publicly accessible pipeline for engineering CRISPR systems enabling much faster optimization for gene editing approaches. Finally, we test our most potent mini-nuclease in human iPSC-derived cardiomyocytes.