MOSAIC enables in situ 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 in situ. Here we describe the development and characterization of the Multiplexing Of Site-specific Alterations for In situ Characterization (MOSAIC) 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 in situ saturation mutagenesis screens of: (1) the BCR-ABL1 fusion gene, successfully identifying known and potentially new imatinib drug-resistance variants; and (2) the IRF1 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 (pegRNA) 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. ### Competing Interest Statement J.K.J. and two other investigators who worked on an NIH award supporting this research, but are not authors on this publication, are co-founders of and have a financial interest in SeQure, Dx, Inc., a company developing technologies for gene editing target profiling. J.K.J. also has, or had during the course of this research, financial interests in several companies developing gene editing technology: Beam Therapeutics, Blink Therapeutics, Chroma Medicine, Editas Medicine, EpiLogic Therapeutics, Excelsior Genomics, Hera Biolabs, Monitor Biotechnologies, Nvelop Therapeutics (f/k/a ETx, Inc.), Pairwise Plants, Poseida Therapeutics, and Verve Therapeutics. J.K.J.'s interests were reviewed and were managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict-of-interest policies. J.K.J. is a co-inventor on various patents and patent applications that describe gene editing and epigenetic editing technologies. L. P. has financial interests in SeQure Dx and Edilytics, Inc. The interests of L.P. were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. J.Y.H has financial interests in Gensaic. K.-C.L. and J.K.J. are presently employees of Arena BioWorks LLC.