AcrVA4 is an anti-CRISPR (Acr) protein that was discovered via STSS (self-targeting spacers search) with further functional TXTL (cell-free transcription-translation) assay. The bioinformatic STSS method finds self-targeting CRISPR spacers in genomic DNA, predicts the type of CRISPR system involved, and obtains information about the targeted sequence. AcrVA4 block Cas12a activity; it inhibits recognition of double-stranded DNA (dsDNA), and drives dimerization of Cas12a. Cas12a (Cpf1) is a class 2 effector protein for the AcrVA subfamily that has high efficiency in genome editing. It possesses a single nuclease domain (RuvC) that is activated upon binding of the crRNA targeting sequence (or spacer) to a complementary single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) target molecule. In addition to gene editing, CRISPR-Cas12a has repurposed, in high eukaryotes, to carry out transcription regulation via a nuclease-dead Cas12a (dCas12a), AcrVAs were adopted to regulate (d)Cas12a activity; AcrVA4 is able to inhibit a dCas12a protein (dLbCas12a) in Saccharomyces cerevisiae. Type V systems fundamentally differ from type II by the domain architecture of their effector proteins. The type II effectors (Cas9) contain two nuclease domains that are each responsible for the cleavage of one strand of the target DNA, with the HNH nuclease inserted inside the RuvC-like nuclease domain sequence. By contrast, the type V effectors (Cas12) only contain a RuvC-like domain that cleaves both strands. Acr proteins have co-evolved with CRISPR-Cas proteins to provide bacteriophage with broad spectrum, or in some cases, highly selective protection from RNA-guided destruction. CRISPR-Cas immune systems are used by certain prokaryotes and archaea to resist the invasion of foreign nucleic acids such as phages or plasmids. Acr proteins are small proteins which are the natural inhibitors for CRISPR-Cas systems; encoded on bacterial and archaeal viruses, they allow the virus to evade host CRISPR-Cas systems. The CRISPR-Cas-mediated adaptive immune response can be divided into three steps, including the acquisition of spacer derived from invading nucleic acids, crRNA processing, and target degradation. Theoretically, Acr proteins could suppress any step to disrupt the CRISPR-Cas system. Acr proteins are diverse with no common sequence or structural motif, and they inhibit a wide range of CRISPR-Cas systems with various inhibition mechanisms. CRISPR-Cas systems are divided into two classes (1 and 2) and six types (class 1: types I, III and IV; class 2: types II, V and VI). Acr families are named for their type and subtype which are numbered sequentially as they are discovered.
Jiyao W et al.(2023). "The conserved domain database in 2023.",