1:Department of Pathology, Massachusetts General Hospital and Harvard Medical School

 Prime editors (PEs) allow for the installation of a broad range of genetic edits. However, their large size makes them more challenging for research and therapeutic use. PE2, the most common PE system, relies on the use of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused to a pentamutant Moloney Murine Leukemia Virus reverse transcriptase (MMLV-RT). Here we report that that the separate expression of nSpCas9 and untethered MMLV-RT can still mediate efficient prime editing in human cells, which suggests that the RT functions in trans. We used this finding to rapidly engineer and test a variety of alternative RT domains, including a ~25% more compact variant of MMLV-RT that lacks the RNAse H domain as well as a bacterial group II intron maturase RT domain, that we engineered in a stepwise process in order to allow for prime editing in human cells. In sum, our findings improve the mechanistic understanding of prime editing, offer a platform for accelerated screening of new PE designs, and provide split and compact PEs with improved flexibility for expression and delivery in human cells.