The iterative cycling of mutation and selection utilized by vertebrate immune systems is a powerful means for generating proteins with diverse properties. In order to apply this approach to the generation of LAGLIDADG homing endonucleases (LHEs) with novel DNA binding and cleavage specificities, we have developed methods to express LHEs as fusion proteins on the surface of cultured B-cells. The surface expressed fusion proteins allow the rapid assessment of the specific binding and cleavage properties of the LHE using flow cytometry, and also can be used to separate populations of cells expressing LHE's with 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 generated by Component 2 (Monnat) and Component 3 (Baker).
In Specific Aim 2, we will integrate surface expressed LHE's into immunoglobulin loci of the DT40 cell line such that they become susceptible to the endogenous somatic hypermutation mechanism(s) operating in DT40 cells. We will then use these LHEhypermutating lines to execute two strategies of iterative mutation/selection to identify novel LHE's with desired binding and cleavage properties. In the first, we will use LHE's from Component 3 (Baker) with pre-optimized DNA/protein interfaces towards target sites, and will attempt to directly select LHE variants able to bind and cleave the predicted target. In the 2nd, we will attempt a base pair-by-base pair migration strategy, beginning with presently available surface expressed LHE scaffolds.
In Specific Aim 3, we will work on further refining and enhancing our methods and technologies for iterative mutation/selection of LHE 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, for use in both flow cytometry and soluble LHE assays. In the second, we will utilize overexpression of proteins involved in somatic hypermutation to incrase the rate of hypermutation and enhance the spectrum of 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 identified here will be passed on to Component 2 (Monnat) and Component 5 (Stoddard) for biochemical and biophysical analysis, with information thus derived incorporated into PSSM matrices for identifying the best engineerable sites by Component 2 (Monnat), and into computational design algorithms developed by Component 3 (Baker).

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Linked Research project Grant (RL1)
Project #
5RL1CA133832-05
Application #
8088051
Study Section
Special Emphasis Panel (ZRR1-SRC (99))
Program Officer
Knowlton, John R
Project Start
2007-09-25
Project End
2013-06-30
Budget Start
2011-07-01
Budget End
2013-06-30
Support Year
5
Fiscal Year
2011
Total Cost
$437,416
Indirect Cost
Name
Seattle Children's Hospital
Department
Type
DUNS #
048682157
City
Seattle
State
WA
Country
United States
Zip Code
98105
Lambert, Abigail R; Hallinan, Jazmine P; Shen, Betty W et al. (2016) Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity. Structure 24:862-73
Thyme, Summer; Song, Yifan (2016) Computational Design of DNA-Binding Proteins. Methods Mol Biol 1414:265-83
Sather, Blythe D; Romano Ibarra, Guillermo S; Sommer, Karen et al. (2015) Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template. Sci Transl Med 7:307ra156
Thyme, Summer; Baker, David (2014) Redesigning the specificity of protein-DNA interactions with Rosetta. Methods Mol Biol 1123:265-82
Baxter, Sarah K; Scharenberg, Andrew M; Lambert, Abigail R (2014) Engineering and flow-cytometric analysis of chimeric LAGLIDADG homing endonucleases from homologous I-OnuI-family enzymes. Methods Mol Biol 1123:191-221
Thyme, Summer B; Boissel, Sandrine J S; Arshiya Quadri, S et al. (2014) Reprogramming homing endonuclease specificity through computational design and directed evolution. Nucleic Acids Res 42:2564-76
Wang, Yupeng; Khan, Iram F; Boissel, Sandrine et al. (2014) Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus. Nucleic Acids Res 42:6463-75
Boissel, Sandrine; Jarjour, Jordan; Astrakhan, Alexander et al. (2014) megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering. Nucleic Acids Res 42:2591-601
Thyme, Summer B; Song, Yifan; Brunette, T J et al. (2014) Massively parallel determination and modeling of endonuclease substrate specificity. Nucleic Acids Res 42:13839-52
Kuhar, Ryan; Gwiazda, Kamila S; Humbert, Olivier et al. (2014) Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks. Nucleic Acids Res 42:e4

Showing the most recent 10 out of 25 publications