User:Luis Andres Casavilca Ramirez/Sandbox 1

Introduction

PduCas13b is a class 2 type 4 from Prevotella buccae. As a member of this protein family, it uses a single effector complexed with a CRISPR-RNA to bind and cleave RNA with its twin HEPN domains, activating a general non-specific RNase activity that degrades any nearby transcripts.(1)⁠ However, PduCas13b is structurally and mechanically different from the other Cas13s, as it has a unique linear organization and different dynamics in target recognition.(2)⁠.

Structure

Though the overall conserved bilobed shape of many Class 2 effectors consisting of a recognition and nuclease lobe is present,(2,3)⁠ Cas13b has a unique domain organization even among Cas13s. (Fig.1). A crystal structure of PbuCas13b complexed with a 36-nt direct repeat sequence and a 5-nt spacer at 1.65 Å provides information of the . There are five domains within the effector structure: two HEPN domains (), two mainly helical domains (), and a Lid domain. In addition, crRNA also has a unique structure, since its direct repeat is at the 3’ end and not at the 5’ end as in the other Cas13s (Fig.1).

HEPNs (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain) are alpha helical domains, many of which are present in RNA maturation systems and related biological conflicts and most of which contain a catalytic Rx4-6H active site. Cas 13b domain is composed of 12 linearly connected alpha helices flexible loop between them, whereas contains nine alpha helices, several short beta strands and beta-hairpin with positively charged residues at the tip. In accordance with the conserved from HEPNs, a Rx4-6H site is present, where active residues locate within both HEPN1 () and HEPN2 () domains.*(all this residues should be shown at the same time, as in the image example) One of the two histidines is thought act as a base, inducing the ribose 2’OH to attack the phosphodiester linkage. The conserved arginine could stabilize the negative oxygens of the transition state, as a similar residue (Lysine) has been shown to carry out such function in RNase A. Alternatively, it could interact with the RNA backbone. The other polar residues, located in between the catalytic histidine and the conserved arginine, are thought to further augment the active site. (2,4)⁠ It is worth noting that, as in most CRISPR associated RNases, nuclease activity at this site is metal-dependent, as target cleavage only occurs in the presence of a certain concentration of Mg2+ and is abolished after addition of EDTA.(5)⁠ This feature contrasts with most HEPN nucleases, where activity is metal-independent. A CRISPR associated RNase, Csx2 from Pyrococcus furious, contains a Zn2+ within its HEPN domain, suggesting a possible role of a divalent cation in further stabilizing the reaction intermediate (2).⁠

A highly conserved inter-domain linker () connects HEPN1 with Helical-1 and spans a highly positive central inner channel, where crRNA lies isolated from the solvent.*(show surface electrostatics of all protein (without crRNA) hiding only the Lid domain, and the highly positively charged inner channel will appear in dark blue. See image example 2) Helical-1 domain makes extensive contacts with the direct repeat of crRNA and minor interface contacts with HEPNs and Lid domains, whereas Helical-2, which is composed of 11 helices, makes extensive contacts with HEPN1 and minor contacts with the extended beta-hairpin of the Lid domain. Between Helical1 and Helical2 domains there is a side channel oppositely oriented from the Lid domain. This second side channel also is positively charged and establishes access to the unbound state crRNA from the solvent.

The Lid domain caps the 3’ end of the direct repeat with two charged (show3’ region and two beta-hairpins of Lid domain). This region has been demonstrated to function as a second nuclease site, involved in pre-crRNA processing. Six conserved residues are predicted to coordinate and process crRNA at this second non-HEPN . (see Image example3). (2)⁠Unlike the HEPN active site, this nuclease site is thought to be metal-independent, since the activity of the equivalent crRNA processing site in Cas13a remains unaffected by the addiction of chelators and there is a formation of a 2′,3′-cyclic phosphate and a 5′-hydroxide on the 5′ and 3′ halves of the crRNA cleavage products, respectively. This last feature is characteristic of metal-independent RNA hydrolysis. (6,7)

cnRNA and Targeting

Application