Discovery and engineering of small SlugCas9 with broad targeting range and high specificity and activity

Abstract

The compact CRISPR/Cas9 system, which can be delivered with their gRNA and a full-length promoter for expression by a single adeno-associated virus (AAV), is a promising platform for therapeutic applications. We previously identified a compact SauriCas9 that displays high activity and requires a simple NNGG PAM, but the specificity is moderate. Here, we identified three compact Cas9 orthologs, Staphylococcus lugdunensis Cas9 (SlugCas9), Staphylococcus lutrae Cas9 (SlutrCas9) and Staphylococcus haemolyticus Cas9 (ShaCas9), for mammalian genome editing. Of these three Cas9 orthologs, SlugCas9 recognizes a simple NNGG PAM and displays comparable activity to SaCas9. Importantly, we generated a SlugCas9-SaCas9 chimeric nuclease, which has both high specificity and high activity. We finally engineered SlugCas9 with mutations to generate a high-fidelity variant that maintains high specificity without compromising on-target editing efficiency. Our study offers important minimal Cas9 tools that are ideal for both basic research and clinical applications.

Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo

“…Engineering of the PAM-interacting domain of SauCas9 reduced its wild-type PAM requirements (5′-NNGRRT-3′) 22 to 5′-NNNRRT 36 , helping to expand its targeting flexibility. More recently, Nme2Cas9 (5′-NNNNCC-3′ PAM) 24 , SauriCas9 (5′-NNGG-3′ PAM) 25 , and SlugCas9 (5′-NNGG-3′ PAM) 37 were discovered as natural compact Cas9s with dinucleotide PAMs and robust editing activity in mammalian cells. To our knowledge, the in vivo utility of SauriCas9 and SlugCas9 with all-in-one AAV vectors is yet to be documented.…”

Compact SchCas9 Recognizes the Simple NNGR PAM

“…We previously identified four SaCas9 orthologs that recognize different PAMs, including NNGG, NNGRR, and NNGGV. [ 15 , 16 ] In this study, by screening a list of 16 SaCas9 orthologs, we identified Cas9s that recognize NNGRRT, NNGRRR, NNGRC, NNGA, and NNGR PAMs, respectively. These PAMs contain invariable G at position 3, G/A at position 4, and less conserved nucleotides at positions 5 and 6, which can be explained by the PI sequence of these Cas9s.…”

“…Numerous CRISPR/Cas9 loci have been sequenced, but only a handful of them have been developed for genome editing. We recently developed four SaCas9 orthologs for mammalian genome editing, [ 15 , 16 ] expanding the targeting scope. In this study, to investigate the diversity of SaCas9 ortholog PAMs, we screened a list of 16 naturally occurring SaCas9 orthologs for genome editing.…”

“…[1] The DSBs are repaired by either non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways, resulting in the desired mutations. [2] We recently developed four SaCas9 orthologs for mammalian genome editing, [15,16] expanding the targeting scope. In this study, to investigate the diversity of SaCas9 ortholog PAMs, we screened a list of 16 naturally occurring SaCas9 orthologs for genome editing.…”

CRISPR Modeling and Correction of Cardiovascular Disease

“…173 Many Cas9 variants that are significantly smaller than SpCas9 have been identified, each with distinct PAM sites. [174][175][176][177][178][179][180][181] The efficacy of packaging smaller variants together with sgRNAs into a single AAV vector has been validated in vivo in a variety of studies. 176,178,[182][183][184] An all-in-one AAV9 vector expressing CjCas9 and a single sgRNA was able to correct a frameshift mutation in DMD mice.…”