Abstract
The broad spectrum of activities displayed by CRISPR-Cas systems has led to biotechnological innovations that are poised to transform human therapeutics. Therefore, the comprehensive characterization of distinct Cas proteins is highly desirable. Here we expand the repertoire of nucleases for mammalian genome editing using the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9). We define functional protospacer adjacent motif (PAM) sequences and variables required for robust and efficient editing in vitro. Expression of holo-St1Cas9 from a single adeno-associated viral (rAAV) vector in the neonatal liver rescued lethality and metabolic defects in a mouse model of hereditary tyrosinemia type I demonstrating effective cleavage activity in vivo. Furthermore, we identified potent anti-CRISPR proteins to regulate the activity of both St1Cas9 and the related type II-A Staphylococcus aureus Cas9 (SaCas9). This work expands the targeting range and versatility of CRISPR-associated enzymes and should encourage studies to determine its structure, genome-wide specificity profile and sgRNA design rules.