Heath Nicholas G, O'Geen Henriette, Halmai Nicole B, Corn Jacob E, Segal David J
Genome Center and Department of Biochemistry and Molecular Medicine, University of California, Davis, Davis, CA, United States.
Integrative Genetics and Genomics, University of California, Davis, Davis, CA, United States.
Front Genome Ed. 2022 Mar 25;4:867390. doi: 10.3389/fgeed.2022.867390. eCollection 2022.
An extensive arsenal of biosensing tools has been developed based on the clustered regularly interspaced short palindromic repeat (CRISPR) platform, including those that detect specific DNA sequences both and in live cells. To date, DNA imaging approaches have traditionally used full fluorescent reporter-based fusion probes. Such "always-on" probes differentiate poorly between bound and unbound probe and are unable to sensitively detect unique copies of a target sequence in individual cells. Herein we describe a DNA biosensor that provides a sensitive readout for such low-copy DNA sequences through proximity-mediated reassembly of two independently optimized fragments of NanoLuc luciferase (NLuc), a small, bright luminescent reporter. Applying this "turn-on" probe in live cells, we demonstrate an application not easily achieved by fluorescent reporter-based probes, detection of individual endogenous genomic loci using standard epifluorescence microscopy. This approach could enable detection of gene edits during editing procedures and should be a useful platform for many other live cell DNA biosensing applications.
基于成簇规律间隔短回文重复序列（CRISPR）平台，已经开发出了大量的生物传感工具，包括那些能够在体外和活细胞中检测特定DNA序列的工具。迄今为止，DNA成像方法传统上使用基于全荧光报告基因的融合探针。这种“一直开启”的探针在结合和未结合的探针之间区分能力较差，并且无法灵敏地检测单个细胞中目标序列的独特拷贝。在此，我们描述了一种DNA生物传感器，它通过接近介导的两个独立优化的NanoLuc荧光素酶（NLuc，一种小型、明亮的发光报告基因）片段的重新组装，为这种低拷贝DNA序列提供灵敏的读数。在活细胞中应用这种“开启式”探针，我们展示了一种基于荧光报告基因的探针不易实现的应用，即使用标准落射荧光显微镜检测单个内源性基因组位点。这种方法能够在基因编辑过程中实现基因编辑的检测，并且应该是许多其他活细胞DNA生物传感应用的有用平台。
