Characterization of homing endonuclease binding and cleavage specificities using yeast surface display SELEX (YSD-SELEX).
LAGLIDADG homing endonucleases (LHEs) are a class of rare-cleaving nucleases that possess several unique attributes for genome engineering applications. An important approach for advancing LHE technology is the generation of a library of design ‘starting points’ through the discovery and characterization of natural LHEs with diverse specificities. However, while identification of natural LHE proteins by sequence homology from genomic and metagenomic sequence databases is straightforward, prediction of corresponding target sequences from genomic data remains challenging. Here, we describe a general approach that we developed to circumvent this issue that combines two technologies: yeast surface display (YSD) of LHEs and systematic evolution of ligands via exponential enrichment (SELEX). Using LHEs expressed on the surface of yeast, we show that SELEX can yield binding specificity motifs and identify cleavable LHE targets using a combination of bioinformatics and biochemical cleavage assays. This approach, which we term YSD-SELEX, represents a simple and rapid first principles approach to determining the binding and cleavage specificity of novel LHEs that should also be generally applicable to any type of yeast surface expressible DNA-binding protein. In this marriage, SELEX adds DNA specificity determination to the YSD platform, and YSD brings diagnostics and inexpensive, facile protein-matrix generation to SELEX.
Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA, USA.|Molecular and Cellular Biology Program, University of Washington, Seattle, WA, USA.|Department of Immunology, University of Washington School of Medicine, Seattle, WA, USA.
Kyle
K
Jacoby
Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA, USA.
Abigail R
AR
Lambert
Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA, USA.|Molecular and Cellular Biology Program, University of Washington, Seattle, WA, USA.|Department of Immunology, University of Washington School of Medicine, Seattle, WA, USA.|Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, USA.
Andrew M
AM
Scharenberg
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D052156:SELEX Aptamer Technique / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; D004720:Endonucleases