Schweitzer Anabel Y, Adams Etowah W, Nguyen Michael T A, Lek Monkol, Isaacs Farren J
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA.
Systems Biology Institute, Yale University, West Haven, CT, USA.
Nat Commun. 2025 May 31;16(1):5061. doi: 10.1038/s41467-025-59653-x.
Base editors enable the direct conversion of target nucleotides without introducing DNA double strand breaks, making them a powerful tool for creating point mutations in a human genome. However, current Cas9-derived base editing technologies have limited ability to simultaneously edit multiple loci with base-pair level precision, hindering the generation of polygenic phenotypes. Here, we test the ability of six Cas12a-derived base editing systems to process multiple gRNAs from a single transcript. We identify base editor variants capable of multiplexed base editing and improve the design of the respective gRNA array expression cassette, enabling multiplexed editing of 15 target sites in multiple human cell lines, increasing state-of-the-art in multiplexing by three-fold in the field of mammalian genome engineering. To reduce bystander mutations, we also develop a Cas12a gRNA engineering approach that directs editing outcomes towards a single base-pair conversion. We combine these advances to demonstrate that both strategies can be combined to drive multiplex base editing with greater precision and reduced bystander mutation rates. Overcoming these key obstacles of mammalian genome engineering technologies will be critical for their use in studying single nucleotide variant-associated diseases and engineering synthetic mammalian genomes.
碱基编辑器能够直接将目标核苷酸进行转换，而不会引入DNA双链断裂，使其成为在人类基因组中产生点突变的强大工具。然而，当前源自Cas9的碱基编辑技术在以碱基对水平的精度同时编辑多个位点方面能力有限，这阻碍了多基因表型的产生。在此，我们测试了六种源自Cas12a的碱基编辑系统处理来自单个转录本的多个gRNA的能力。我们鉴定出了能够进行多重碱基编辑的碱基编辑器变体，并改进了相应gRNA阵列表达盒的设计，从而能够在多种人类细胞系中对15个靶位点进行多重编辑，在哺乳动物基因组工程领域将多重编辑的现有技术水平提高了三倍。为了减少旁观者突变，我们还开发了一种Cas12a gRNA工程方法，该方法可将编辑结果导向单一碱基对转换。我们结合这些进展来证明这两种策略可以结合起来，以更高的精度和更低的旁观者突变率驱动多重碱基编辑。克服哺乳动物基因组工程技术的这些关键障碍对于其用于研究单核苷酸变异相关疾病和构建合成哺乳动物基因组至关重要。
