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 CRISPR separator. Inspired by this observation, we design short, AT-rich synthetic separators (synSeparators) 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.

中文翻译：

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使用 CRISPR 阵列分离器增强 Cas12a 多基因调控


VA Cas12a 蛋白可以处理其 CRISPR 阵列，这一功能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 阵列常常表现出不可预测的性能。在这里，我们报告 Cas12a 阵列性能对 gRNA 间隔区的 GC 含量非常敏感，因为高 GC 间隔区会损害下游 gRNA 的活性。我们分析天然存在的 CRISPR 阵列，并观察到天然重复序列总是包含富含 AT 的片段，用于分隔 gRNA，我们将其称为CRISPR 分隔符。受这一观察的启发，我们设计了短的、富含 AT 的合成分离器 ( synSeparators )，成功消除了 gRNA 之间的破坏性影响。我们进一步证明了使用包含 synSeparator 的阵列增强了人类细胞中七个内源基因的同时激活。这些结果阐明了天然 CRISPR 阵列先前未被充分探索的功能，并展示了受自然启发的工程解决方案如何改善哺乳动物细胞中的多基因控制。