One More Piece Down to Solve the III-A CRISPR Puzzle

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats) loci function in tandem with the adjacent operon of Cas (CRISPR associated) proteins to provide prokaryotes with an RNA-based adaptive immune system against foreign genetic elements (reviewed in Ref. [1]). The centerpiece of all CRISPR-Cas systems is a large ribonucleoprotein interference complex that uses a single-stranded crRNA (CRISPR RNA) for nucleic acid targeting and allows for the subsequent degradation [2]. The ribonucleoproteins come in different flavors for each of the three major CRISPR types (types I–III) and the multiple subtypes therein. They have been under intense structural and mechanistic characterization. CRISPR-Cas systems are stringently classified into one of three types based on the presence of the signature protein in their Cas operon [3] and less formally classified by the general architecture of their interference complexes. Type I systems utilize the Cascade (CRISPR associated complex for antiviral defense), a multi-subunit complex composed of five different proteins and a crRNA. TheCascade employs the associated crRNA to locate and invade matching double-stranded DNA and for the target degradation by the signature nuclease–helicase enzyme Cas3 [2,3]. Electronmicroscopyand crystal structures of the Cascade in both apoform and single-stranded DNA bound forms are now available [4–7]. In addition, the crystal structure of Cas3 bound to a single-stranded DNA substrate has been determined [8]. Therefore, the frontier of the field has shifted toward understanding mechanistic details regarding the Cascade-mediated recruitment of Cas3 and the subsequent substrate degradation. Type II systems use the single large signature protein Cas9 to carry out crRNAguided targeting and degradation of double-stranded DNA; a trans-encoded RNA assists the process [9,10]. The simplicity of this CRISPR type enabled