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

Abstract

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.

Keywords: CRISPR; Cas proteins; anti-CRISPR; gene editing; synthetic biology.

FIGURE 1

CRISPR-Cas-mediated pathways for DNA degradation. (A) Type I. Cas6 protein cleaves the pre-crRNA to generate the mature crRNA. Cascade-crRNA binds the DNA and recruits Cas3 that, first, induces a nick in the non-target strand (NTS), 7–11 nt downstream of PAM. Then, Cas3 degrades the nicked strand in an ATP-dependent manner. Afterward, Cas3 binds, cleaves, and degrades the target strand (TS) in the same way. Genes enclosed in a dotted line are not required by all subtypes. (B) Type V-A. Cas12a protein processes the pre-crRNA into mature crRNA and uses the only RuvC domain (in the NUC lobe) to cleave both strands of the substrate DNA, which provokes DNA degradation.

FIGURE 2

The structure of type I-F system and its interactions with AcrIF proteins. In the absence of AcrI proteins, the Csy-crRNA complex (made of Cas6f, Cas7.1f-7.6f, Cas5f–Cas8f, and the crRNA) binds the target DNA sequence. The resulting crRNA-DNA heteroduplex recruits Cas3 that carries out DNA cleavage. AcrI proteins prevent DNA degradation in two ways. AcrIF1, AcrIF2, AcrIF6, and AcrIF8-F10 hinder the interaction of Cascade with the DNA by binding different subunits of the Csy complex. In contrast, AcrIF3 blocks the function of Cas3. The precise inhibitory mechanism of AcrIF4 is still unknown.

FIGURE 3

The inhibitory mechanism of AcrVA proteins. AcrVA1 and AcrVA4 prevent Cas12a-crRNA from binding dsDNA. AcrVA1 works by mimicking PAM to disrupt the communication among PI and WED (in Cas12a NUC lobe), and the DNA. Besides, AcrVA1 can also truncate the crRNA. In the complex Cas12a-crRNA-AcrVA4, AcrVA4 dimer drives two Cas12a-crRNAs to form a butterfly structure that prevents the structural change required for crRNA-DNA heteroduplex formation and catalytic cleavage activation. AcrVA5 is very distinct from the other two AcrVA proteins. In order to abolish Cas12a, AcrVA5 works as an acetyltransferase and transfers the acetyl group from acetyl-CoA to LbCas12a K595. Acetylated Cas12a can no longer interact with PAM. The sequence in orange represents the crRNA spacer.

FIGURE 4

AcrVA1-mediated multiple-input synthetic gene circuit. The genetic network in the figure expresses the therapeutic factor IFNγ, in a controlled way, upon detection of tumor-relevant signals in HEK293T cells. The chimeric activator dCas12a-miniVPR-crRNA (where miniVPR is an activation domain) regulates the synthesis of IFNγ, the circuit output. dCas12a-miniVPR is split into two parts: the N-terminal dCas12a (N-dCas12a) and the C-terminal dCas12a fused to miniVPR (C-dCas12a-miniVPR). Both parts are expressed under inducible promoters: the hypoxia-inducible promoter (hypoxia can signal the presence of a tumor) leads C-dCas12a-miniVPR synthesis, whereas the endogenous RRM2 promoter (ribonucleotide reductase regulatory subunit M2) drives the production of N-dCas12a in response to TFs (transcription factors) whose occurrence is due to a tumor. crRNA molecules, which bind dCas12a-miniVPR, are constitutively transcribed. AcrVA1 amount is controlled by the antibiotic doxycycline (Dox). When two tumor-relevant signals (hypoxia and TFs) appear in the cells, IFNγ are produced and can be detected by the ELISA assay. AcrVA1 is used to modulate the activity of dCas12a-miniVPR-crRNA and, hence, the level of the output signal.