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B - Gene Targeting and Gene Correction -> Gene Targeting and Gene Correction (Basic development of novel technologies for genome editing, with or without site-specific endonuclease. Abstracts focused on specific disease applications should be submitted to the suitable tissue/disease category)

MOSAIC Enables In Situ Saturation Mutagenesis of Genes and Optimization of CRISPR Prime Editor Activities in Human Cells

Type: Oral Abstract Session

Presentation Details
Session Title: Late-Breaking Abstracts I
Location: Salon H
Start Time: 5/17/2022 8:45
End Time: 5/17/2022 9:00

CRISPR prime editing enables tremendous versatility and precision for creating a broad range of genetic edits in human cells. Here we describe the development of the Multiplexing Of Site-specific Alterations for In situ Characterization (MOSAIC) method, a non-viral PCR-based strategy that enables the rapid installation of thousands of defined prime editing-mediated genetic edits in pooled fashion. Using MOSAIC, we were able to rapidly and easily perform pooled in situ saturation mutagenesis screens of the BCR-ABL1 gene translocation and the IRF1 untranslated region (UTR), re-confirming known imatinib drug-resistance coding sequence mutations in BCR-ABL1 and non-coding regulatory elements in the IRF1 UTR. Furthermore, we also leveraged MOSAIC to enable high-throughput, pooled screening of hundreds of prime editing guide RNA (pegRNA) designs for any given targeted modification of interest. Using MOSAIC, we screened more than 18,000 pegRNA designs and identified optimized pegRNAs for 89 different genomic target modifications. The results of these screens reveal the lack of simple rules for pegRNA design and demonstrate the requirement to perform experimental optimization, a process that is now greatly simplified and can be practiced by any scientist using MOSAIC. In sum, MOSAIC provides an important and novel technology that should facilitate the deployment of CRISPR prime editing for pooled screens and optimize pegRNAs for a wide variety of different research and therapeutic applications.

Jonathan Y. Hsu1,2,3,4, Kin Chung Lam2,3,4, Justine Y. Shih2,3,4, Luca Pinello2,3,4, Keith Joung2,3,4

1Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA,2Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA,3Center for Cancer Research and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA,4Department of Pathology, Harvard Medical School, Boston, MA
 J.Y. Shih: None.

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