Curtin Medical School, Curtin University, Bentley, WA, Australia.
Curtin Health Innovation Research Institute, Curtin University, Bentley, WA, Australia.
Nat Commun. 2022 May 31;13(1):3023. doi: 10.1038/s41467-022-30598-9.
The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. However, this promise has long been limited by the technical challenges involved in genetic engineering. Recent advances in gene editing have bypassed some of these challenges but they are still far from ideal. Here we use FuncLib to computationally design Cas9 enzymes with substantially higher donor-independent editing activities. We use genetic circuits linked to cell survival in yeast to quantify Cas9 activity and discover synergistic interactions between engineered regions. These hyperactive Cas9 variants function efficiently in mammalian cells and introduce larger and more diverse pools of insertions and deletions into targeted genomic regions, providing tools to enhance and expand the possible applications of CRISPR-based gene editing.
改变活细胞基因组的能力是理解基因如何影响生物功能的关键，对于有目的地修饰生物系统也至关重要。然而，这一承诺长期以来一直受到遗传工程中所涉及的技术挑战的限制。最近基因编辑方面的进展绕过了其中的一些挑战，但它们仍远非理想。在这里，我们使用 FuncLib 计算设计了 Cas9 酶，使其具有更高的供体非依赖性编辑活性。我们使用与酵母细胞存活相关的遗传回路来定量 Cas9 活性，并发现了工程化区域之间的协同相互作用。这些高活性 Cas9 变体在哺乳动物细胞中有效发挥作用，并在靶向基因组区域中引入更大和更多样化的插入和缺失，为增强和扩展基于 CRISPR 的基因编辑的可能应用提供了工具。
