Visualization of phage DNA degradation b.y a type I CRISPR-Cas system at the single-cell level

作     者：Jingwen Guan Xu Shi Roberto Burgos Lanying Zeng 

作者机构：Department of Biochemistry and Biophysics Texas A&M University College Station TX 77843 USA Center for Phage Technology Texas A&M University College Station TX 77843 USA Molecular and Environmental Plant Sciences Texas A&M University College station TX 77843 USA 

出 版 物：《Frontiers of Electrical and Electronic Engineering in China》 (中国电气与电子工程前沿（英文版）)

年 卷 期：2017年第5卷第1期

页      面：67-75页

核心收录：

学科分类：12[管理学] 1201[管理学-管理科学与工程(可授管理学、工学学位)] 08[工学] 

基　　金：supported by the National Institutes of Health 

主　　题：bacteriophage lambda CRISPR-Cas fluorescence microscopy single-cell analysis type I CRISPR 

摘      要：Background： The CRISPR-Cas system is a widespread prokaryotic defense system which targets and cleaves invasive nucleic acids, such as plasmids or viruses. So far, a great number of studies have focused on the components and mechanisms of this system, however, a direct visualization of CRISPR-Cas degrading invading DNA in real-time has not yet been studied at the single-cell level. Methods： In this study, we fluorescently label phage lambda DNA in vivo, and track the labeled DNA over time to characterize DNA degradation at the single-cell level. Results： At the bulk level, the lysogenization frequency of cells harboring CRISPR plasmids decreases significantly compared to cells with a non-CRISPR control. At the single-cell level, host cells with CRISPR activity are unperturbed by phage infection, maintaining normal growth like uninfected cells, where the efficiency of our antilambda CRISPR system is around 26%. During the course of time-lapse movies, the average fluorescence ofinvasive phage DNA in cells with CRISPR activity, decays more rapidly compared to cells without, and phage DNA is fully degraded by around 44 minutes on average. Moreover, the degradation appears to be independent of cell size or the phage DNA ejection site suggesting that Cas proteins are dispersed in sufficient quantities throughout the cell. Conclusions： With the CRISPR-Cas visualization system we developed, we are able to examine and characterize how a CRISPR system degrades invading phage DNA at the single-cell level. This work provides direct evidence and improves the current understanding on how CRISPR breaks down invading DNA.