Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells

• Published in: Nature communications Vol. 10; no. 1; p. 194
• Main Authors: Nakamura, Muneaki, Srinivasan, Prashanth, Chavez, Michael, Carter, Matthew A., Dominguez, Antonia A., La Russa, Marie, Lau, Matthew B., Abbott, Timothy R., Xu, Xiaoshu, Zhao, Dehua, Gao, Yuchen, Kipniss, Nathan H., Smolke, Christina D., Bondy-Denomy, Joseph, Qi, Lei S.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 14-01-2019; Nature Publishing Group; Nature Portfolio
• Subjects: 13/106; 13/31; 14; 14/63; 42/109; 631/337/4041; 631/61/338/552; Bacteriophages - genetics; Cell Culture Techniques; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR-Cas Systems - genetics; Crystal defects; Crystals; Eukaryotic Cells; Gene Editing - methods; Gene Regulatory Networks - genetics; Genetic Vectors - genetics; Genome editing; HEK293 Cells; Humanities and Social Sciences; Humans; Image resolution; Induced Pluripotent Stem Cells; Intravital Microscopy - methods; Lentivirus - genetics; Low temperature; Luminescence; Microscopy, Fluorescence - methods; multidisciplinary; Nanotechnology; Nanowires; Perovskites; Phages; Phase transitions; Science; Science (multidisciplinary); Spectroscopic analysis; Spectroscopy; Temperature effects; Time-Lapse Imaging - methods; Transduction, Genetic - methods; Transfection - methods; Transition temperature
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-018-08158-x

Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate “write-protected” cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.
Anti-CRISPR proteins derived from phage can abrogate CRISPR activity. The authors repurpose these molecules for demonstrating genomic write-protection and pre-programmed gene expression circuits.