Towards sequential conjugation-assisted laboratory evolution (SCALE) of Cas nucleases

DNA endonucleases such as the CRISPR-associated enzymes Cas9 and Cas12a constitute powerful molecular tools with a wide range of applications. To facilitate optimization of these enzymes, we investigated a fullyin vivoselection system for laboratory evolution based on plasmid conjugation. In the proposed method, DNA cleavage promotes plasmid transfer to other cells, allowing cleaving variants to spread through the bacterial culture via consecutive conjugations. We have investigated whether the conjugation system of the F plasmid ofE. coliis suitable for laboratory evolution of Cas nucleases. Through a series of conjugation experiments, we found (i) that the F plasmid can spread through bacterial cultures, (ii) that the FinOP conjugation inhibition system is a potential nuclease target as it can prevent transfer of non-cleaving variants (counterselection), and (iii) that a trimmed version of the F plasmid allows for consecutive conjugations. Overall, we were able to show that in many regards, conjugation can indeed be used as means ofin vivolaboratory evolution. We discuss the potential of the here presented concept for a yet to be developed laboratory evolution platform.