gene-digital-circuits-based-on-crispr-cas-systems-anti-crispr

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the ...

Tianjin University

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins), a prokaryotic RNA-mediated adaptive immune system, has been repurposed for gene editing and synthetic gene circuit construction both in bacterial and eukaryotic cells. In the last years, the emergence of the anti-CRISPR proteins (Acrs), which are natural OFF-switches for CRISPR-Cas, has provided a new means to control CRISPR-Cas activity and promoted a further development of CRISPR-Cas-based biotechnological toolkits. In this review, we focus on type I and type V-A anti-CRISPR proteins. We first narrate Acrs discovery and analyze their inhibitory mechanisms from a structural perspective. Then, we describe their applications in gene editing and transcription regulation. Finally, we discuss the potential future usage-and corresponding possible challenges-of these two kinds of anti-CRISPR proteins in eukaryotic synthetic gene circuits.

Types I and V Anti-CRISPR Proteins: From Phage Defense to Eukaryotic Synthetic Gene Circuits.

Type V-A anti-CRISPR proteins (AcrVAs) represent the response from phages to the CRISPR-Cas12a prokaryotic immune system. CRISPR-Cas12a was repurposed, in high eukaryotes, to carry out gene editing and transcription regulation, the latter via a nuclease-dead Cas12a (dCas12a). Consequently, AcrVAs were adopted to regulate (d)Cas12a activity. However, the usage of both dCas12a-based transcription factors and AcrVAs in the yeast  has not been explored. In this work, we show that, in the baker's yeast, two dCas12a proteins (denAsCas12a and dLbCas12a) work both as activators (upon fusion to a strong activation domain) and repressors, whereas dMbCa12a is nonfunctional. The activation efficiency of dCas12a-ADs manifests a dependence on the number of crRNA binding sites, whereas it is not directly correlated to the amount of crRNA in the cells. Moreover, AcrVA1, AcrVA4, and AcrVA5 are able to inhibit dLbCa12a in yeast, and denAsCas12a is only inhibited by AcrVA1. However, AcrVA1 performs well at high concentration only. Coexpression of two or three AcrVAs does not enhance inhibition of dCas12a(-AD), suggesting a competition between different AcrVAs. Further, AcrVA4 significantly limits gene editing by LbCas12a. Overall, our results indicate that dCas12a:crRNA and AcrVA proteins are highly performant components in  synthetic transcriptional networks.

Lifang Yu

School of Pharmaceutical Science and Technology

Tianjin University

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins