Anti-CRISPR-mediated control of gene editing and synthetic circuits 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.

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

“…Notably, genomic material withdrawal from chassis organism bestows circumstances in which desired synthesis reactions are achievable at high rates . Thus, such features enable prevailing utilization of CFPS systems, which are also complementary to in vivo expression for various applications such as biocatalyst development (Rolf, Rosenthal, & Lütz, 2019), complex product fabrication (Chi, Wang, Li, Ren, & Huang, 2015), disease detection (Soltani, Davis, Ford, Nelson, & Bundy, 2018), glycoprotein synthesis (Jaroentomeechai et al, 2018), prototyping minimal cells (Yue, Zhu, & Kai, 2019), synthetic gene networks (Dubuc et al, 2019), as well as protein engineering (Hong, Kwon, & Jewett, 2014;Venkat, Chen, Gan, & Fan, Sheng, Lei, Yuan and Feng (2017) Functional CRISPR gene editing toolkit Marshall et al (2018) Pathway/network prototyping UDP-N-acetylglucosamine and UDP-GlcNAc pathway Zhou et al (2010) CRISPR-mediated gene activation and repression of the reporter and endogenous genes in mammalian cells Nakamura et al (2019) Protein engineering E. coli strain mutant for release factor 1 allows turning UAG termination codon into a sense codon for site-specific incorporation of nonnatural amino acids into proteins for selective conjugation with different biomolecules Adachi et al (2019) Incorporation of uAA AzF allows for site-specific mono and dipegylation of T4 Lysozyme Wilding et al (2018) Incorporating optimized nonnatural amino acids site-specifically, the feasibility of conjugating a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug by para-azidomethyl-L-phenylalanine (pAMF) to the tumor-specific, Her2-binding IgG Trastuzumab Zimmerman et al (2014) Biosensing Cheomogenic detection of estrogenic endocrine disruptors (hERb) in human blood and urine Salehi et al (2018) Glycoengineering Site-specific controlled glycosylation of proteins (glycoproteins) using E. coli extracts enriched for oligosaccharyltransferases and lipidlinked oligosaccharides Jaroentomeechai et al (2018) Site-directed incorporation of AzF, as well as ppropargyloxyphenylalanine from Sf21 insect cell, extracts for engineered O-glycosylation site of EPO Zemella et al (2018) Protein evolution: ribosome display Protein-tRNA-ribosome-mRNA complex for affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin Mattheakis, Bhatt and Dower (1994) Protein evolution: mRNA display Protein-puromycin-mRNA complex Roberts...…”

Anti‐CRISPRs: The natural inhibitors for CRISPR‐Cas systems

“…Some of the type II Acr proteins can also inactivate dCas9‐based genome editing technologies by blocking dCas9 binding to the target DNA. Several studies have revealed that AcrIIA4 can significantly inhibit gene regulation by CRISPRi, CRISPRa and targeted DNA demethylation in human cells . Moreover, optogenetic controlled AcrIIA4 and inducible AcrIIA4 can modulate Cas9‐mediated genome or epigenome editing …”

Allosteric Inhibition of CRISPR-Cas9 by Bacteriophage-derived Peptides

“…Acrs can be adapted to regulate CRISPR-Cas activities in bacteria [34], yeast [35] and mammalian cells [29,31,34,[36][37][38]. Biosensor [39] and synthetic circuits [40] can be devised based on Acr-coupled CRISPR-Cas systems. Moreover, Acrs can be harnessed to enable the temperatureresponsive [41] and optogenetic [42] control of CRISPR-Cas activity.…”