Gene

Molecular biology

Biology

Cas9

Genome editing

Prime (order theory)

Virology

Operating system

Computer science

Computational biology

Modular design

Genetics

Genome

Nuclease

Mathematics

Reverse transcriptase

Combinatorics

Guide RNA

Nature Biotechnology

Delivery and optimization of prime editors (PEs) have been hampered by their large size and complexity. Although split versions of genome-editing tools can reduce construct size, they require special engineering to tether the binding and catalytic domains. Here we report a split PE (sPE) in which the Cas9 nickase (nCas9) remains untethered from the reverse transcriptase (RT). The sPE showed similar efficiencies in installing precise edits as the parental unsplit PE3 and no increase in insertion–deletion (indel) byproducts. Delivery of sPE to the mouse liver with hydrodynamic injection to modify β-catenin drove tumor formation with similar efficiency as PE3. Delivery with two adeno-associated virus (AAV) vectors corrected the disease-causing mutation in a mouse model of type I tyrosinemia. Similarly, prime editing guide RNAs (pegRNAs) can be split into a single guide RNA (sgRNA) and a circular RNA RT template to increase flexibility and stability. Compared to previous sPEs, ours lacks inteins, protein–protein affinity modules and nuclease-sensitive pegRNA extensions, which increase construct complexity and might reduce efficiency. Our modular system will facilitate the delivery and optimization of PEs. A split prime editor simplifies delivery.

A split prime editor with untethered reverse transcriptase and circular RNA template