Journal
NATURE COMMUNICATIONS
Volume 13, Issue 1, Pages -Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41467-022-30514-1
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Funding
- National Institutes of Health (NIH) [R01HL157714]
- Cancer Prevention and Research Institute of Texas [CPRIT RP180734]
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This study introduces a novel method for multiplex gene editing in human cells using CRISPR array architecture and RNA polymerase III promoter, enabling simultaneous editing of multiple disease-related genomic loci.
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. 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 the authors describe drive-and-process CRISPR array architectures for multiplex base-editing and multiplex prime-editing in human cells.
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