Quantification of evolved DNA-editing enzymes at scale with DEQSeq.

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.

Discovery and characterization of novel Cre-type tyrosine site-specific recombinases for advanced genome engineering.

Tyrosine-type site-specific recombinases (Y-SSRs) are versatile tools for genome engineering due to their ability to mediate excision, integration, inversion and exchange of genomic DNA with single nucleotide precision. The ever-increasing need for sophisticated genome engineering is driving efforts to identify novel SSR systems with intrinsic properties more suitable for particular applications. In this work, we develop a systematic computational workflow for annotation of putative Y-SSR systems and apply this pipeline to identify and characterize eight new naturally occurring Cre-type SSR systems. We test their activity in bacterial and mammalian cells and establish selectivity profiles for the new and already established Cre-type SSRs with regard to their ability to mutually recombine their target sites. These data form the basis for sophisticated genome engineering experiments using combinations of Y-SSRs in research fields including advanced genomics and synthetic biology. Finally, we identify putative pseudo-sites and potential off-targets for Y-SSRs in the human and mouse genome. Together with established methods for altering the DNA-binding specificity of this class of enzymes, this work should facilitate the use of Y-SSRs for future genome surgery applications.