Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses

Abstract

The combination of yeast surface display and flow cytometric analyses and selections is being used with increasing frequency to alter specificity of macromolecular recognition, including both protein-protein and protein-nucleic acid interactions. Here we describe the use of yeast surface display and cleavage-dependent flow cytometric assays to increase the specificity of an engineered meganuclease. The re-engineered meganuclease displays a significantly tightened specificity profile, while binding its cognate target site with a slightly lower, but still sub-nanomolar affinity. When incorporated into otherwise identical megaTAL protein scaffolds, these two nucleases display significantly different activity and toxicity profiles in cellulo. The structural basis for reprogrammed DNA cleavage specificity was further examined via high-resolution X-ray crystal structures of both enzymes. This analysis illustrated the altered protein-DNA contacts produced by mutagenesis and selection, that resulted both in altered readout of those based and a necessary reduction in DNA binding affinity that were necessary to improve specificity across the target site. The results of this study provide an illustrative example of the potential (and the challenges) associated with the use of surface display and flow cytometry for the retargeting and optimization of enzymes that act on nucleic acid substrates in a sequence-specific manner.

CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice

“…Raw reads were pre-processed using tools from the Galaxy suite 99 or using a previously described pipeline. 100 Briefly, reads were trimmed using BBDuk ( https://jgi.doe.gov/data-and-tools/bb-tools/ ), Trimmomatic, 101 and cutadapt 102 to remove adaptor contaminants and low-quality regions (Q < 30) at the 3′ and 5′ ends; any remaining reads shorter than 20 nt were discarded. Trimmed reads were mapped to the HBV sequence using Bowtie 2 103 and exported for further analysis.…”

A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets

“…Similarly to other display methods, its principle is based on cycles of naïve protein library exposure, selection, and enrichment of yeast clones with desired properties. Yeast display has proven to be an effective method for developing, , improving, and altering activities , of proteins for research, therapeutic, and biotechnology applications. The unprecedented power of the technique, together with its relative ease of use and reasonable cost, has made it popular in many laboratories around the world.…”