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

• Published in: Nature communications Vol. 13; no. 1; pp. 2771 - 13
• Main Authors: Yuan, Qichen, Gao, Xue
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 19.05.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 45/22; 45/23; 45/29; 45/91; 631/208; 631/337/4041/3196; 631/61; Arrays; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; Constraining; CRISPR; CRISPR-Cas Systems - genetics; DNA-directed RNA polymerase; Editing; Gene Editing; Gene expression; Gene loci; Genomics; gRNA; Humanities and Social Sciences; Humans; Medical research; multidisciplinary; Multiplexing; RNA polymerase; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Transfer - genetics; Science; Science (multidisciplinary); Therapeutic applications; Transfer RNA; tRNA Cys
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-30514-1

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.