The repurposing of type I-E CRISPR-Cascade for gene activation in plants

Joshua K Young,Pedro Navarro,Becca Vickroy,Spencer Jones,Lijuan Wang,Rodolphe Barrangou,Stephen L Gasior

doi:10.1038/s42003-019-0637-6

Joshua K Young, Pedro Navarro + Show 5 more

Open Access

https://doi.org/10.1038/s42003-019-0637-6

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Abstract

CRISPR-Cas systems are robust and facile tools for manipulating the genome, epigenome and transcriptome of eukaryotic organisms. Most groups use class 2 effectors, such as Cas9 and Cas12a, however, other CRISPR-Cas systems may provide unique opportunities for genome engineering. Indeed, the multi-subunit composition of class 1 systems offers to expand the number of domains and functionalities that may be recruited to a genomic target. Here we report DNA targeting in Zea mays using a class 1 type I-E CRISPR-Cas system from S. thermophilus. First, we engineer its Cascade complex to modulate gene expression by tethering a plant transcriptional activation domain to 3 different subunits. Next, using an immunofluorescent assay, we confirm Cascade cellular complex formation and observe enhanced gene activation when multiple subunits tagged with the transcriptional activator are combined. Finally, we examine Cascade mediated gene activation at chromosomal DNA targets by reprogramming Zea mays cells to change color.

Highlights

CRISPR-Cas systems are robust and facile tools for manipulating the genome, epigenome and transcriptome of eukaryotic organisms
To minimize impact on CRISPRassociated complex for antiviral defense (Cascade) complex activity and ensure nuclear localization signal (NLS) recognition, the structure of the type I-E Cascade complex from E. coli (Eco) was referenced[26] and associated termini likely to be not buried in the SthCascade complex were selected as NLS attachment points
CasA, CasD, and CasE were selected for cold binding factor 1 (CBF1) attachment as they represent monomeric subunits in the SthCascade complex and simplify experiments aimed at comparing gene activation potential with Cas[9]

Summary

Introduction

CRISPR-Cas systems are robust and facile tools for manipulating the genome, epigenome and transcriptome of eukaryotic organisms. Additional accessory protein domains are tethered to the amino and carboxyl termini or guide RNA (gRNA) bringing novel functionalities to the Cas-gRNA complex Such fusions have been used to upregulate or downregulate gene expression[8], deaminate DNA9, modify the epigenome[10], and visualize genomic loci[11]. Until very recently[12], only class 2 CRISPR-Cas systems, comprised of a single effector protein (type II-Cas[9] and type V-Cas12)[13], have been adopted as eukaryotic genome editing tools. The processive single stranded DNA exonuclease associated with type I systems, Cas[3], provides a new approach for the introduction of large chromosomal deletions adjacent to Cascade DNA targets[13]. We report the first use of type I CRISPR-

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