TY - JOUR TI - Enhanced Cas12a multi-gene regulation using a CRISPR array separator AU - Magnusson, Jens P AU - Rios, Antonio Ray AU - Wu, Lingling AU - Qi, Lei S A2 - Wade, Joseph T A2 - Weigel, Detlef A2 - Beisel, Chase A2 - Wade, Joseph T VL - 10 PY - 2021 DA - 2021/09/09 SP - e66406 C1 - eLife 2021;10:e66406 DO - 10.7554/eLife.66406 UR - https://doi.org/10.7554/eLife.66406 AB - 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. KW - CRISPR KW - Cas12a KW - multiplexed gene regulation KW - CRISPR array KW - separator KW - GC content KW - Guide RNA design KW - gene activation KW - CRISPRa KW - Cpf1 KW - Cas13d KW - mammalian cells KW - human cells JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -