MOSAIC enables saturation mutagenesis of genes and CRISPR prime editing guide RNA optimization in human cells.

CRISPR prime editing offers unprecedented versatility and precision for the installation of genetic edits . Here we describe the development and characterization of the Multiplexing Of Site-specific Alterations for Characterization ( ) method, which leverages a non-viral PCR-based prime editing method to enable rapid installation of thousands of defined edits in pooled fashion. We show that MOSAIC can be applied to perform saturation mutagenesis screens of: (1) the fusion gene, successfully identifying known and potentially new imatinib drug-resistance variants; and (2) the untranslated region (UTR), re-confirming non-coding regulatory elements involved in transcriptional initiation. Furthermore, we deployed MOSAIC to enable high-throughput, pooled screening of hundreds of systematically designed prime editing guide RNA ( ) constructs for a large series of different genomic loci. This rapid screening of >18,000 pegRNA designs identified optimized pegRNAs for 89 different genomic target modifications and revealed the lack of simple predictive rules for pegRNA design, reinforcing the need for experimental optimization now greatly simplified and enabled by MOSAIC. We envision that MOSAIC will accelerate and facilitate the application of CRISPR prime editing for a wide range of high-throughput screens in human and other cell systems.