Jacoby Kyle, Lambert Abigail R, Scharenberg Andrew M
Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA, USA.
Molecular and Cellular Biology Program, University of Washington, Seattle, WA, USA.
Nucleic Acids Res. 2017 Feb 17;45(3):e11. doi: 10.1093/nar/gkw864.
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.
LAGLIDADG归巢内切酶（LHEs）是一类罕见的切割核酸酶，在基因组工程应用中具有几个独特的属性。推进LHE技术的一个重要方法是通过发现和表征具有不同特异性的天然LHEs来生成一个设计“起点”文库。然而，虽然从基因组和宏基因组序列数据库中通过序列同源性鉴定天然LHE蛋白很简单，但从基因组数据中预测相应的靶序列仍然具有挑战性。在这里，我们描述了一种我们开发的通用方法来规避这个问题，该方法结合了两种技术：LHEs的酵母表面展示（YSD）和指数富集配体系统进化（SELEX）。使用在酵母表面表达的LHEs，我们表明SELEX可以产生结合特异性基序，并通过生物信息学和生化切割分析相结合的方法鉴定可切割的LHE靶标。我们将这种方法称为YSD-SELEX，它代表了一种简单快速的第一原理方法，用于确定新型LHEs的结合和切割特异性，该方法也应普遍适用于任何类型的可在酵母表面表达的DNA结合蛋白。在这种结合中，SELEX为YSD平台增加了DNA特异性测定，而YSD为SELEX带来了诊断和廉价、便捷的蛋白质-基质生成。
