Staphylococcus aureus Cas9 is a multiple-turnover enzyme

Abstract

Cas9 nuclease is the key effector of type II CRISPR adaptive immune systems found in bacteria. The nuclease can be programmed by a single guide RNA (sgRNA) to cleave DNA in a sequence-specific manner. This property has led to its widespread adoption as a genome editing tool in research laboratories and holds great promise for biotechnological and therapeutic applications. The general mechanistic features of catalysis by Cas9 homologs are comparable; however, a high degree of diversity exists among the protein sequences, which may result in subtle mechanistic differences. S. aureus (SauCas9) and especially S. pyogenes (SpyCas9) are among the best-characterized Cas9 proteins and share ∼17% sequence identity. A notable feature of SpyCas9 is an extremely slow rate of reaction turnover, which is thought to limit the amount of substrate DNA cleavage. Using in vitro biochemistry and enzyme kinetics, we directly compare SpyCas9 and SauCas9 activities. Here, we report that in contrast to SpyCas9, SauCas9 is a multiple-turnover enzyme, which to our knowledge is the first report of such activity in a Cas9 homolog. We also show that DNA cleaved with SauCas9 does not undergo any detectable single-stranded degradation after the initial double-stranded break observed previously with SpyCas9, thus providing new insights and considerations for future design of CRISPR/Cas9-based applications.

A catalogue of biochemically diverse CRISPR-Cas9 orthologs

“…5b). Analysis of Sau Cas9 target cleavage produced almost entirely blunt-ended products as shown earlier 40 ( Fig. 5b and Supplementary Fig.…”

CRISPR/Cas brings plant biology and breeding into the fast lane

“…Recently, GT frequencies in Arabidopsis could be improved by the use of the Cas9 orthologue SaCas9 instead of SpCas9 [33 ]. This surprising effect is supported by biochemical data that SaCas9, in contrast to SpCas9, has a multiple turnover activity, allowing frequent cleavage of the target substrate [34]. However, the most important improvement for GT in Arabidopsis was the use of the egg-cell-specific EC1 transcriptional cassette for nuclease expression that has been shown to be efficient in NHEJ-based editing before its use for GT in plants [35].…”

Delivery Approaches for Therapeutic Genome Editing and Challenges

“…In addition, SaCas9 reagents, similar to other Cas homologs, are commercially available to enable ease of their direct delivery as preformed sgRNA-Cas9 RNPs for potent cleavage activity at endogenous sites in cells. In contrast to SpCas9, the advantages of SaCas9 nuclease are its compact cargo size (~3.3 kb) and its multiple-turnover enzymatic activity, which has a faster rate of substrate release that causes no additional detectable nuclease activity to occur upon DNA cleavage [ 57 ]. The drawback of the SaCas9 is its limited targeting range due to its relatively long PAM sequence requirement.…”