Abstract

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. There are several approaches known by now to obtain differentiated CRISPR systems with new properties. Some known smaller Cas9s are an alternative, but often lack robust activity or specificity and frequently recognize longer PAMs. Here, we present four previously uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. We apply protein engineering to generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.