Targeted Transcriptional Modulation with Type I CRISPR-Cas Systems in Human Cells

The CRISPR revolution has highlighted bacterial adaptive immune systems as a rich and largely unexplored frontier for discovery of new genome engineering technologies. In particular, Cas9, a class 2 CRISPR-Cas systems, has been widely applied for targeting DNA sequences in eukaryotic genomes. However, the great diversity of CRISPR systems existing throughout the natural world has not yet been explored for potential genome engineering tools in human cells. Here, we report DNA-targeting and transcriptional control with class 1 CRISPR-Cas systems. Specifically, we repurposed the Cascade effector complex from type I variants of class 1 CRISPR-Cas systems, the most prevalent CRISPR loci in nature. Cascade targets double-stranded DNA via a multi-component protein complex and a single CRISPR RNA (crRNA). We first designed codon-optimized mammalian expression cassettes for each Cascade subunit from the E. coli strain K12 (termed EcoCascade). Following transfection of human cells, we validated expression of each subunit, proper nuclear localization, and Cascade complex formation. We demonstrated programmable crRNA-mediated targeting of specific loci in the human genome via ChIP-seq analysis. By tethering transactivation domains to Cascade, we modulated the expression of multiple targeted chromosomal genes in human cells, including >3,000-fold IL1RN activation comparable to activation with dCas9-based effectors. Transcriptome-wide analysis by RNA-seq showed highly specific crRNA-dependent activation of the targeted gene. To generalize this approach to other Cascade complexes, we applied our understanding of the model type I-E EcoCascade to the type I-B CRISPR-Cas system of L. monocytogenes Finland_1998 (termed LmoCascade). Following tethering of transactivation domains to LmoCascade, we showed endogenous gene activation in human cells with almost all tested crRNAs. Additionally, we tethered the KRAB effector to LmoCascade and demonstrated robust targeted transcriptional repression at endogenous loci. This study expands the toolbox for engineering mammalian genomes and establishes Cascade as a novel CRISPR-based technology for targeted mammalian gene regulation.