基于和先导编辑的多重 CRISPR 驱动-过程阵列。
Multiplex base- and prime-editing with drive-and-process CRISPR arrays.
机构信息
Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, USA.
Department of Bioengineering, Rice University, Houston, TX, USA.
出版信息
Nat Commun. 2022 May 19;13(1):2771. 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.
当前的碱基编辑和 Prime 编辑技术缺乏高效的策略来同时编辑多个基因组位点，限制了它们在复杂基因组和多基因疾病中的应用。在这里，我们描述了用于人类细胞中多路碱基编辑（MBE）和多路 Prime 编辑（MPE）的驱动和处理（DAP）CRISPR 阵列架构。我们利用 tRNA 作为 RNA 聚合酶 III 启动子来驱动串联组装的 tRNA 指导 RNA（gRNA）阵列的表达，其中单个 gRNA 被细胞内源性 tRNA 加工机制释放。我们为 DAP 阵列工程设计了一种 75nt 的人半胱氨酸 tRNA（hCtRNA），实现了多达 31 个位点的 MBE 和多达 3 个位点的 MPE。通过应用 MBE 或 MPE 元件通过腺相关病毒（AAV）和慢病毒进行传递，我们证明了多个与疾病相关的基因组位点的同时编辑。我们的工作简化了单个阵列上 gRNA 的表达和处理，并为生物医学研究和治疗应用建立了高效的 MBE 和 MPE 策略。
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