Engineered CRISPR prime editors with compact, untethered reverse transcriptases

The CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use this Split-PE system to rapidly identify and engineer more compact prime editor architectures that also broaden the types of RTs used for prime editing. A split prime editor architecture facilitates screening and engineering of improved variants.

Automated summary

The CRISPR prime editor PE2, composed of nSpCas9 and MMLV-RT, functions efficiently in human cells. The Split-PE system allows for the identification and engineering of more compact prime editor architectures that broaden the types of RTs used for prime editing. A split prime editor architecture facilitates screening and engineering of improved variants.