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

TLDR

Repurposed CRISPR‑Cas molecules enable broad genomic editing and gene regulation in prokaryotes and eukaryotes, and the recent discovery of phage‑derived anti‑CRISPR proteins that abrogate natural CRISPR activity expands the ability to build synthetic CRISPR‑mediated circuits. The study characterizes a panel of anti‑CRISPR molecules to counteract CRISPR‑mediated gene activation and repression of reporter and endogenous genes across various cell types. The authors characterized a panel of anti‑CRISPR molecules and tested their ability to inhibit CRISPR‑mediated gene activation and repression in reporter and endogenous genes across multiple cell types. The study shows that pre‑engineered cells with anti‑CRISPR molecules are resistant to gene editing, enabling write‑protected cells, and that anti‑CRISPRs can control CRISPR‑based gene regulation circuits—including a pulse generator in mammalian cells—demonstrating their broad utility for synthetic systems in eukaryotic cells.

Abstract

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.