Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Nat Chem Biol. 2024 Feb;20(2):211-220. doi: 10.1038/s41589-023-01431-1. Epub 2023 Sep 28.
Monitoring environmental DNA can track the presence of organisms, from viruses to animals, but requires continuous sampling of transient sequences from a complex milieu. Here we designed living sentinels using Bacillus subtilis to report the uptake of a DNA sequence after matching it to a preencoded target. Overexpression of ComK increased DNA uptake 3,000-fold, allowing for femtomolar detection in samples dominated by background DNA. This capability was demonstrated using human sequences containing single-nucleotide polymorphisms (SNPs) associated with facial features. Sequences were recorded with high efficiency and were protected from nucleases for weeks. The SNP could be determined by sequencing or in vivo using CRISPR interference to turn on reporter expression in response to a specific base. Multiple SNPs were recorded by one cell or through a consortium in which each member recorded a different sequence. Sentinel cells could surveil for specific sequences over long periods of time for applications spanning forensics, ecology and epidemiology.
环境 DNA 监测可以追踪从病毒到动物等生物体的存在，但需要从复杂环境中持续采样短暂的序列。在这里，我们使用枯草芽孢杆菌设计了活体哨兵，用于在匹配到预编码目标后报告 DNA 序列的摄取。ComK 的过表达将 DNA 摄取增加了 3000 倍，使得在以背景 DNA 为主的样本中能够检测到飞摩尔级别的 DNA。这一功能通过使用与人面部特征相关的单核苷酸多态性 (SNP) 相关的人类序列进行了演示。序列以高效率被记录下来，并在数周内免受核酸酶的破坏。通过测序或体内使用 CRISPR 干扰来确定 SNP，以响应特定碱基打开报告基因表达。单个细胞或通过一个联盟可以记录多个 SNP，其中每个成员记录一个不同的序列。哨兵细胞可以在很长一段时间内监测特定序列，适用于法医学、生态学和流行病学等多个领域。
