Characterization ofStreptococcus uberisCas9 (SuCas9) – a Type II-A Ortholog Functional in Human Cells

Type II CRISPR-Cas9 RNA-guided nucleases are commonly used for genome engineering. To date, all characterized Cas9-based genome editors, including the widely used SpCas9, have limitations such as their relatively large size and restriction of targets flanked by a specific PAM sequence. Here, we biochemically characterized more compact SpCas9 ortholog, SuCas9, from Streptococcus uberis , a bacterium inhabiting the mammary glands of dairy cattle. SuCas9 recognizes a novel 5′-NNAAA-3′ PAM, efficiently cleaves DNA in vitro , and is active in human cells. The study of SuCas9 has the potential to expand the range of applications of CRISPR-Cas9 enzymes in medicine and biotechnology.