Fig. 4

DEQSeq identifies favorable Cas12f-derived miniABEs. a Workflow overview. Evolved Cas12f-ABEs are cloned together with a unique molecular identifier (UMI) into a vector containing three target sites (yellow boxes). The number of transformed bacteria is determined by counting colonies on appropriate agar plates. Calculated from the transformation efficiency, a defined number of transformed bacteria is cultured. Cas12f-ABE expression is induced by the addition of L-arabinose, which may result in base editing of the target sites on the plasmid. The plasmid DNA is isolated, and the region containing the Cas12f-ABE gene, the UMI, and the target sites is excised and sequenced with nanopore sequencing. The sequences of the UMIs are computationally clustered based on similarity which results in groups of sequences corresponding to the different variants. From these groups, the base editing events on the different target sites are counted and accurate Cas12f-ABE sequences are generated. b DEQSeq screen of Cas12f-ABE on three target sites. UMI-clusters containing evolved Cas12f-ABEs are indicated as blue points and WT control clusters are indicated in black. Clusters that were characterized are highlighted