A CRISPR RNA Is Closely Related With the Size of the Cascade Nucleoprotein Complex

Abstract

The currently known prokaryotic adaptive immune system against mobile genetic elements is based on clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated (Cas) proteins and the transcribed short CRISPR RNA (crRNA) molecule form a heterologous ribonucleoprotein complex that neutralizes invading foreign nucleic acids, wherein the crRNA molecule base-pairs with the exogenous genetic elements. In the ribonucleoprotein complexes of the type I CRISPR system, a helical backbone of six identical subunits is commonly found. However, it is not clear how this ribonucleoprotein complex is assembled and what is the determinant factor for its size. We elucidated the crystal structure of the Csy3 subunit of the type I-F ribonucleoprotein complex from Zymomonas mobilis (ZmCsy3), in which seven ZmCsy3 protomers in the asymmetric unit form a molecular helix that is part of a filamentous structure in the entire crystal system. This ZmCsy3 helical structure is remarkably similar to the crRNA-bound hexameric Csy3 backbone from Pseudomonas aeruginosa, with conserved interactions between neighboring subunits. The monomeric ZmCsy3 in solution is transformed into different oligomeric states depending on the added crRNAs. These results suggest that a crRNA and Csy3 subunit play a determinant role in the stepwise formation of the functional Cascade ribonucleoprotein complex and the recruitment of other subunits, and crRNA functions as a molecular ruler for determining the size of the Cascade silencing complex.

Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells

“…1b). Each Csy3 binds to 6-nt of the crRNA spacer with the precise number of Csy3 subunits determined by the length of the crRNA spacer 56 (from 14 to 50-nt), resulting in 3-9 copies of Csy3 57 . Csy4 binds to the crRNA 3′ hairpin structure and is responsible for pre-crRNA maturation (Fig.…”

Type I-F CRISPR-PAIR platform for multi-mode regulation to boost extracellular electron transfer in Shewanella oneidensis

“…Cas6f combines with crRNA 3’ hairpin structure and mediates pre-crRNA maturation. Cas7 functioning as several copies binds to the protospacer, serving as the backbone of the cascade complex ( Gleditzsch et al., 2016 ; Gu et al., 2019 ). Compared to the standalone dCas9, more than one effector for activation or inhibition fused to Cas7 can be recruited to the targets, allowing stronger transcriptional regulation ( Figure 1 ) ( Cady et al., 2012 ; Chen et al., 2020 ).…”

DNA interference is controlled by R-loop length in a type I-F1 CRISPR-Cas system

“…Cascade complexes in the type I-E and type I-F2 can incorporate spacers, which length differ from the original spacer length found in WT complex [52,55,59,60]. Recently, RNA length was shown to govern the oligomeric state of isolated Zymomonas mobilis Cas7f1 [61]. We wanted to determine the range of spacer lengths, which can be incorporated into stable type I-F1 Cascade complex.…”