...One way to reconstitute a split protein like Cas9 is to use split inteins. Split inteins are a pair of naturally...challenge that the field has overcome with split AAVs. In general split AAVs break a large transgene in two ..., mediates protein trans-splicing, similar to an intron in pre-mRNA splicing. In 2016, Fine et al developed...Staphylococcus aureus Cas9 (SaCas9, 1053 amino acids) displayed cleavage activity in mammalian cells. SaCas9 produced...sensitive method, they found that SaCas9 did not display higher levels of off-target activity than SpCas9...length, allowing them to be packaged in AAV.
Split AAV approaches
While finding smaller Cas9 orthologs...developed a proof-of-concept split intein SpCas9 which had modest editing rates in HEK-293T cells when compared...