Department of Bioengineering, Stanford University, Stanford, United States.
Department of Chemical and Systems Biology, Stanford University, Stanford, United States.
Elife. 2021 Sep 9;10:e66406. doi: 10.7554/eLife.66406.
The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple guide RNA (gRNAs). Here, we report that Cas12a array performance is hypersensitive to the GC content of gRNA spacers, as high-GC spacers can impair activity of the downstream gRNA. We analyze naturally occurring CRISPR arrays and observe that natural repeats always contain an AT-rich fragment that separates gRNAs, which we term a . Inspired by this observation, we design short, AT-rich synthetic separators () that successfully remove the disruptive effects between gRNAs. We further demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously underexplored feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.
V-A 型 Cas12a 蛋白可以对其 CRISPR 阵列进行加工，这一特性对于多重基因编辑和调控非常有用。然而，由于多个向导 RNA（gRNA）之间的干扰，CRISPR 阵列的性能往往难以预测。在这里，我们报告 Cas12a 阵列的性能对 gRNA 间隔区的 GC 含量高度敏感，因为高 GC 间隔区会损害下游 gRNA 的活性。我们分析了自然发生的 CRISPR 阵列，并观察到天然重复序列总是包含一个富含 AT 的片段，将 gRNA 隔开，我们称之为. 受此观察结果的启发，我们设计了短的、富含 AT 的合成分隔物（），成功消除了 gRNA 之间的干扰效应。我们进一步证明，在含有 synSeparator 的阵列中，可增强对人细胞中七个内源性基因的同时激活。这些结果阐明了自然 CRISPR 阵列中一个以前未被充分探索的特征，并展示了如何借鉴自然启发的工程解决方案来改善哺乳动物细胞中的多基因控制。
