Quantification of evolved DNA-editing enzymes at scale with DEQSeq

Abstract

We introduce DEQSeq, a nanopore sequencing approach that rationalizes the selection of favorable genome editing enzymes from directed molecular evolution experiments. With the ability to capture full-length sequences, editing efficiencies, and specificities from thousands of evolved enzymes simultaneously, DEQSeq streamlines the process of identifying the most valuable variants for further study and application. We apply DEQSeq to evolved libraries of Cas12f-ABEs and designer-recombinases, identifying variants with improved properties for future applications. Our results demonstrate that DEQSeq is a powerful tool for accelerating enzyme discovery and advancing genome editing research.

Engineering spacer specificity of the Cre/loxP system

“…For all three libraries we detected increased band intensities representative of the recombined plasmid product, indicating the enrichment of Cre variants with increased activity (Figure 2C ). To quantitatively investigate a large number of individual clones in the library, we performed DNA Editing Quantification Sequencing (DEQSeq), a high-throughput Nanopore sequencing method that enables the characterization of thousands of DNA editing enzyme variants on multiple target sites ( 30 ). We selected a total of ∼5000 random variants from the three libraries and quantified recombination activity of each of the variants on all four sites ( loxP , loxSE1 , loxSE2 and loxSE3 ) at an average sequencing depth of >1000× reads per variant.…”

“…Once activity on the given site was achieved, the libraries were enriched for active variants with three rounds of low induction and high-fidelity amplification (Herculase II Fusion DNA Polymerase, Agilent) resulting in increased activity of all libraries compared to Cre wt (Figure 2C ). The DEQseq method ( 30 ) was then used for high-throughput evolution of the activity of individual recombinase variants from each library. The three libraries of active variants were barcoded via amplification, pooled together and then cloned into vectors for the DEQseq protocol.…”