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

Qichen Yuan and Xue Gao ()
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Qichen Yuan: Rice University
Xue Gao: Rice University

Nature Communications, 2022, vol. 13, issue 1, 1-13

Abstract: 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.

Date: 2022
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DOI: 10.1038/s41467-022-30514-1

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