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.

Directed evolution studies of a thermophilic Type II-C Cas9

“…Cas9 may be further divided into three subtypes depending on the sequences of Cas9 and the association with proteins involved in spacer acquisition (Chylinski, Le Rhun, & Charpentier, 2013; Mir, Edraki, Lee, & Sontheimer, 2018; Shmakov et al, 2017). The Type II-A and the Type II-B CRISPR–Cas systems contain Csn2 and Cas4, respectively, which are hypothesized spacer acquisition factors, whereas the Type II-C lacks both (Chylinski et al, 2013; Shmakov et al, 2017).…”

“…Among the thousands of Cas9s detectable in the published prokaryote genomes, nearly half belong to Type II-C (Chylinski et al, 2013; Mir et al, 2018; Shmakov et al, 2017). Initial research suggests that Type II-C Cas9s have weaker activities than those of Type II-A Cas9s (Ma, Harrington, O’Connell, Zhou, & Doudna, 2015).…”

“…However, recent works reveal that Type II-C Cas9s are much more diverse than Type II-A Cas9 and possess unique qualities that are worthy further exploration. Type II-C Cas9s are small in size, which makes in vivo delivery using a viral vector easier for genome engineering (Kim et al, 2017; Mir et al, 2018; Tsui et al, 2017). Both the PAMs and the required guide-target base pairing for Type II-C Cas9s are longer than those of other subtypes, which make them inherently more specific (Mir et al, 2018).…”

“…Type II-C Cas9s are small in size, which makes in vivo delivery using a viral vector easier for genome engineering (Kim et al, 2017; Mir et al, 2018; Tsui et al, 2017). Both the PAMs and the required guide-target base pairing for Type II-C Cas9s are longer than those of other subtypes, which make them inherently more specific (Mir et al, 2018). Genome editing in mammalian cells with three Type II-C Cas9s indeed showed a low off-target activity (Esvelt et al, 2013; Harrington et al, 2017; Hou et al, 2013; Kim et al, 2017; Lee, Cradick, & Bao, 2016).…”

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

“…More recently, Campylobacter jejuni and Neisseria meningitidis Cas9s from the type II-C 15 CRISPRCas systems have been added to this repertoire 16,17 .…”