The iterative cycling of mutation and selection utilized by vertebrate immune systems is a powerfulmeans for generating proteins with diverse properties. In order to apply this approach to the generation ofLAGLIDADG homing endonucleases (LHEs) with novel DNA binding and cleavage specificities, we havedeveloped methods to express LHEs as fusion proteins on the surface of cultured B-cells. The surfaceexpressed fusion proteins allow the rapid assessment of the specific binding and cleavage properties ofthe LHE using flow cytometry, and also can be used to separate populations of cells expressing LHE'swith different specificities. Based on these data, we propose the following Specific Aims:
In Specific Aim 1, we will generate and characterize new surface expressed LHE scaffolds based on novel LHE's generatedby Component 2 (Monnat) and Component 3 (Baker).
In Specific Aim 2, we will integrate surfaceexpressed LHE's into immunoglobulin loci of the DT40 cell line such that they become susceptible to theendogenous somatic hypermutation mechanism(s) operating in DT40 cells. We will then use these LHEhypermutatinglines to execute two strategies of iterative mutation/selection to identify novel LHE's withdesired binding and cleavage properties. In the first, we will use LHE's from Component 3 (Baker) withpre-optimized DNA/protein interfaces towards target sites, and will attempt to directly select LHE variantsable to bind and cleave the predicted target. In the 2nd, we will attempt a base pair-by-base pair migrationstrategy, beginning with presently available surface expressed LHE scaffolds.
In Specific Aim 3, we willwork on further refining and enhancing our methods and technologies for iterative mutation/selection ofLHE variants. In one part of this aim, we will work with Component 2 (Monnat) and Component 5(Stoddard) on developing an increased sensitivity cleavage assay based on quantum dot nanosensors, foruse in both flow cytometry and soluble LHE assays. In the second, we will utilize overexpression ofproteins involved in somatic hypermutation to incrase the rate of hypermutation and enhance the spectrumof mutations to include a higher rate of insertions and deletions.The output of this aim directly feeds back to the NGEC LHE design cycle as well, as variants identifiedhere will be passed on to Component 2 (Monnat) and Component 5 (Stoddard) for biochemical andbiophysical analysis, with information thus derived incorporated into PSSM matrices for identifying the bestengineerable sites by Component 2 (Monnat), and into computational design algorithms developed byComponent 3 (Baker).
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