Type II-C CRISPR-Cas9 Biology, Mechanism, and Application

Abstract

Genome editing technologies have been revolutionized by the discovery of prokaryotic RNA-guided defense system called CRISPR-Cas. Cas9, a single effector protein found in type II CRISPR systems, has been at the heart of this genome editing revolution. Nearly half of the Cas9s discovered so far belong to the type II-C subtype but have not been explored extensively. Type II-C CRISPR-Cas systems are the simplest of the type II systems, employing only three Cas proteins. Cas9s are central players in type II-C systems since they function in multiple steps of the CRISPR pathway, including adaptation and interference. Type II-C CRISPR systems are found in bacteria and archaea from very diverse environments, resulting in Cas9s with unique and potentially useful properties. Certain type II-C Cas9s possess unusually long PAMs, function in unique conditions (e.g., elevated temperature), and tend to be smaller in size. Here, we review the biology, mechanism, and applications of the type II-C CRISPR systems with particular emphasis on their Cas9s.

Figure 1

Characteristics of type II-C CRISPR-Cas systems. (A) A typical type II-C CRISPR locus contains a tracrRNA, three cas genes, and the CRISPR array. The spacer sequences in the array are depicted as diamonds, repeat sequences are shown as squares, and the internal promoters are shown as arrows. (B) The domain architecture of type II-C Cas9s. The individual domains are colored separately and labeled. (C) A phylogenetic tree of in vitro validated type II-C Cas9s. The reported PAMs and the protein sizes of Cas9s are also listed. Cas9s validated for mammalian editing are also marked as Y (Yes) or N (No).

Figure 2

Structures of representative type II-C Cas9s and their sgRNAs. (A) The predicted secondary structures of sgRNAs from NmeCas9, GeoCas9, and CjeCas9 are shown. Secondary structures were predicted using mFold. The secondary structure of CjeCas9 as seen in the crystal structure is also shown. (B) From left to right: crystal structures of CjeCas9:sgRNA:DNA, apo AnaCas9, and overlay of CjeCas9 and AnaCas9. The individual domains are colored and are labelled separately. The structures are depicted as cartoons, and were rendered in PyMol using coordinates from pdb entries 4OGE and 5X2H.