清脆的
原位
生物传感器
荧光素酶
计算生物学
DNA
生物
分子生物学
化学
遗传学
生物化学
基因
转染
有机化学
作者
Nicholas G. Heath,David J. Segal
出处
期刊:Methods in molecular biology
日期:2024-01-01
卷期号:: 285-299
标识
DOI:10.1007/978-1-0716-3766-1_19
摘要
To date, CRISPR-based DNA targeting approaches have typically used fusion proteins between full fluorescent reporters and catalytically inactive Cas9 (dCas9) for imaging rather than detection of endogenous genomic DNA sequences. A promising alternative strategy for DNA targeting is the direct biosensing of user-defined sequences at single copy with single-cell resolution. Our recently described DNA biosensing approach using a dual fusion protein biosensor comprised of two independently optimized fragments of NanoLuc luciferase (NLuc) directionally fused to dCas9 paired with user-defined single-guide RNAs (sgRNAs) could allow users to sensitively detect unique copies of a target sequence in individual living cells using common laboratory equipment such as a microscope or a luminescence-equipped microplate reader. Here we describe a protocol for using such a DNA biosensor noninvasively in situ.
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