No abstract

Prime editing leverages Cas proteins fused to reverse transcription enzymes and prime editing guide RNAs (pegRNAs) to programmably engineer diverse genetic alterations without DNA double strand breaks or exogenous donor templates. Prime editing is powerful, but the process of pegRNA design remains a daunting challenge that limits its widespread adoption by the community. Although a number of computational pegRNA design tools have been developed, none so far contain the right combination of features needed for rapid and systematic design of pegRNAs for variant engineering and high-throughput genetic screens. Here, we describe Prime Editing Guide Generator (PEGG): a computational pipeline for rapid design of pegRNAs and ‘sensor’ pegRNA libraries. PEGG is a user-friendly Python package that generates and visualizes pegRNAs based on a list of input mutations and integrates user-defined properties like variable length of primer binding site and reverse transcription template regions, and outputs ready-to-order pegRNA oligos and libraries containing optimized sequence adapters for pooled cloning. PEGG (including documentation) can be accessed athttps://pegg.readthedocs.io.