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. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a “GG” dinucleotide PAM similar to SpyCas9. Protein engineering 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.
Highlights
Streptococcus pyogenes (Spy) Cas[9] has potential as a component of gene therapeutics for incurable diseases
Previously uncharacterized Cas9s from Staphylococcus hyicus (Shy), Staphylococcus lugdunensis (Slu), Staphylococcus microti (Smi), and Staphylococcus pasteuri (Spa), and found three recognized a favorable “NNGG” protospacer adjacent motif (PAM) (Supplementary Fig. 1)
Mouse studies with LNPs and AAV-mediated gene therapy in non-human primate (NHP) show the potential of these additions to the CRISPR toolbox
Summary
Streptococcus pyogenes (Spy) Cas[9] has potential as a component of gene therapeutics for incurable diseases. Effective delivery of CRISPR-Cas systems to targeted cells and tissues is crucial for successful in vivo genome editing. For this purpose, recombinant adeno-associated virus (rAAV) vectors and lipid-nanoparticles (LNPs) are among the most prevalent and promising technologies[9]. Because packaging into rAAV vectors is limited to ~5 kb, Cas[9] proteins smaller than SpyCas[9] are desirable to enable packaging of DNA encoding both Cas[9] and sgRNA into one rAAV (“all-in-one-AAV”) particle This limitation is exacerbated for the larger multidomain-Cas-nuclease-based systems for base editing, prime editing, or CRISPRi/a10–14. Smaller Cas[9] proteins are of keen interest in the field
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
