The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9

Abstract

Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). AceCas9 depends on a 5′-NNNCC-3′ PAM and is more efficient in cleaving negative supercoils than relaxed DNA. Kinetic as well as in vivo activity assays reveal that AceCas9 achieves optimal activity when combined with a guide RNA containing a 24-nucleotide complementarity region. The cytosine-specific, DNA topology-sensitive, and extended guide-dependent properties of AceCas9 may be explored for specific genome editing applications.

Directed evolution studies of a thermophilic Type II-C Cas9

“…The tracrRNA of AceCas9 is predicted to form two stem-loops. The last stem-loop as well as the linker between stem-loop 2 and stem-loop 3 are longer than those of other known Cas9s (Tsui et al, 2017) (Fig. 1).…”

“…3). In this method, survival of the bacterial cells transformed with both a ccdB- and an AceCas9-encoding plasmids is linked to the activity of AceCas9 (Tsui et al, 2017). Successful cleavage of the ccdB plasmid by AceCas9 removes the toxicity to cells and thus their survival in the presence of arabinose.…”

“…The tracrRNA forms an extensive interface with the Cas9, whereas the crRNA is associated with the enzyme complex through a long base-paired region with the tracrRNA (Anders et al, 2014; Garcia-Doval & Jinek, 2017; Hirano, Gootenberg, et al, 2016; Hirano, Nishimasu, et al, 2016; Huai et al, 2017; Jiang et al, 2016; Jiang, Zhou, Ma, Gressel, & Doudna, 2015; Jinek et al, 2014; Nishimasu et al, 2015; Yamada et al, 2017). The tracrRNA and crRNA can be covalently linked at the base-paired region to form a single guide RNA (sgRNA) (Esvelt et al, 2013; Hou et al, 2013; Jinek et al, 2012; Ran et al, 2015; Tsui, Hand, Duboy, & Li, 2017) (Fig. 1).…”

“…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).…”

“…AceCas9 consists of the two well-conserved nuclease domains, HNH and RuvC, responsible for cleaving the target and the nontarget strand of the target nucleic acid, respectively, the PAM interacting domain (PID) that recognizes its PAM sequence, and the REC domain that recognizes the DNA–RNA heteroduplex (Tsui et al, 2017) (Fig. 1).…”