1/20/2024 0 Comments Flowjo x edit a keywprd![]() Cas12a can catalyze the maturation of its gRNAs and has been used for multiplex gene knockouts, transcriptional regulations, and genetic perturbations 15, 16, 17, 18, 19, 20, 21. Although pooling multiple gRNAs is a straightforward method 12, 13, 14, it is not feasible when viral deliveries are needed for in vivo applications. Because the Cas9 used in those editors cannot mature its gRNAs from a single array and existing Cas9-based multiplex strategies require large expression constructs that cause delivery burdens 10, 11. However, the multiplex editing ability of BE and PE is limited. The ability to simultaneously edit multiple genomic loci with BEs or PEs would enable the study of complex functional genomics and the treatment of polygenic diseases. A point mutation in the Lamin A gene that causes the Hutchinson–Gilford progeria syndrome in mice 8, applying PE in human cardiomyocytes to correct the exon 51 deletion mutation in the dystrophin gene that causes Duchenne muscular dystrophy 9.Recently, both base editing (BE) and prime editing (PE) have been used for research and applications in a variety of cell types and animal models 7, e.g., using BE to correct the C Prime editors contain a nCas9 (H840A) tethered to an engineered reverse transcriptase (RT) programmed by prime editing gRNAs (pegRNAs) that encode the desired editing information for targeted insertions, deletions, or installation of all types of point mutations 6. C by CGBEs 4, and dual-deaminase base editors 5.A by cytosine base editors (CBEs) 1, A.DNA base editors are composed of deaminases fused to catalytically impaired nickase Cas9 (nCas9, D10A) and have enabled efficient base-pair conversions, including C ![]() 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.īase editors and prime editors are high-precision genome editing tools that can be programmed to alter the desired context of the genome in living cells, without causing DNA double-strand breaks or requiring DNA donors 1, 2, 3. 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. 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. 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. Here, we describe drive-and-process (DAP) CRISPR array architectures for multiplex base-editing (MBE) and multiplex prime-editing (MPE) in human cells. Current base- and prime-editing technologies lack efficient strategies to edit multiple genomic loci simultaneously, limiting their applications in complex genomics and polygenic diseases. ![]()
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