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Multiplex base- and prime-editing with drive-and-process CRISPR arrays

Author

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  • Qichen Yuan

    (Rice University)

  • Xue Gao

    (Rice University
    Rice University
    Rice University)

Abstract

Current base- and prime-editing technologies lack efficient strategies to edit multiple genomic loci simultaneously, limiting their applications in complex genomics and polygenic diseases. Here, we describe drive-and-process (DAP) CRISPR array architectures for multiplex base-editing (MBE) and multiplex prime-editing (MPE) in human cells. We leverage tRNA as the RNA polymerase III promoter to drive the expression of tandemly assembled tRNA-guide RNA (gRNA) arrays, of which the individual gRNAs are released by the cellular endogenous tRNA processing machinery. We engineer a 75-nt human cysteine tRNA (hCtRNA) for the DAP array, achieving up to 31-loci MBE and up to 3-loci MPE. By applying MBE or MPE elements for deliveries via adeno-associated virus (AAV) and lentivirus, we demonstrate simultaneous editing of multiple disease-relevant genomic loci. Our work streamlines the expression and processing of gRNAs on a single array and establishes efficient MBE and MPE strategies for biomedical research and therapeutic applications.

Suggested Citation

  • Qichen Yuan & Xue Gao, 2022. "Multiplex base- and prime-editing with drive-and-process CRISPR arrays," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-30514-1
    DOI: 10.1038/s41467-022-30514-1
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    1. Hongzhi Zeng & Qichen Yuan & Fei Peng & Dacheng Ma & Ananya Lingineni & Kelly Chee & Peretz Gilberd & Emmanuel C. Osikpa & Zheng Sun & Xue Gao, 2023. "A split and inducible adenine base editor for precise in vivo base editing," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    2. Qichen Yuan & Hongzhi Zeng & Tyler C. Daniel & Qingzhuo Liu & Yongjie Yang & Emmanuel C. Osikpa & Qiaochu Yang & Advaith Peddi & Liliana M. Abramson & Boyang Zhang & Yong Xu & Xue Gao, 2024. "Orthogonal and multiplexable genetic perturbations with an engineered prime editor and a diverse RNA array," Nature Communications, Nature, vol. 15(1), pages 1-12, December.

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