Staphylococcus aureus Cas9 (SauCas9) is a less well-characterized homolog. Sp圜as9 is a monomeric protein that can be programmed withĪ single guide RNA (sgRNA) to induce sequence-specific double-stranded (ds) breaks in DNA ( Jinek et al. Of the diverse families of Cas nucleases, Streptococcus pyogenes Cas9 (Sp圜as9) is the best-characterized and most widely used. 2016 Chen and Doudna 2017 Garcia-Doval and Jinek 2017 Karvelis et al. Our understanding of CRISPR/Cas systems is progressing rapidly, in part drivenīy the excitement about adapting them for use in research, biotechnology, and human therapy ( Wang et al. ![]() Previous Section Next Section INTRODUCTIONĬlustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) constitute sequence-basedĪdaptive immunity in bacteria and archaea. We also show that DNA cleaved with SauCas9 does not undergo any detectable single-stranded degradation after the initial double-strandedīreak observed previously with Sp圜as9, thus providing new insights and considerations for future design of CRISPR/Cas9-based Sp圜as9, SauCas9 is a multiple-turnover enzyme, which to our knowledge is the first report of such activity in a Cas9 homolog. Using in vitroīiochemistry and enzyme kinetics, we directly compare Sp圜as9 and SauCas9 activities. A notable feature of Sp圜as9 isĪn extremely slow rate of reaction turnover, which is thought to limit the amount of substrate DNA cleavage. pyogenes (Sp圜as9) are among the best-characterized Cas9 proteins and share ∼17% sequence identity. The general mechanistic features of catalysis by Cas9 homologs are comparable however, a high degree of diversity existsĪmong the protein sequences, which may result in subtle mechanistic differences. This property has led to its widespread adoptionĪs a genome editing tool in research laboratories and holds great promise for biotechnological and therapeutic applications. The nuclease can be programmedīy a single guide RNA (sgRNA) to cleave DNA in a sequence-specific manner. ![]() Cas9 nuclease is the key effector of type II CRISPR adaptive immune systems found in bacteria.
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