Non-identity-mediated CRISPR-bacteriophage interaction mediated via the Csy and Cas3 proteins.
-
Csy2 and Csy4 are strictly required for crRNA accumulation in vivo, whereas Csy1 and Csy3 are not
"proteins Csy4 and Csy2 are essential for small CRISPR RNA (crRNA) production in vivo, while the Csy1 and Csy3 proteins are not absolutely required for production of these small RNAs"
-
The PA14 Yersinia-subtype locus was reported not to confer resistance to phage DMS3, acting instead on biofilm formation
"the Yersinia-subtype CRISPR region of Pseudomonas aeruginosa strain UCBPP-PA14 plays no detectable role in viral immunity but instead is required for bacteriophage DMS3-dependent inhibition of biofilm formation"
RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions.
-
Csy1-4 assemble into a 350 kDa crRNA-containing surveillance complex that promotes guide-target hybridization
"the Csy proteins (Csy1-4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences"
-
Target recognition initiates at a seed sequence at the crRNA spacer 5' end
"Target recognition is enthalpically driven and localized to a "seed sequence" at the 5' end of the CRISPR RNA spacer"
Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR RNA.
-
Repeat cleavage is a prerequisite for assembly of the Csy effector complex
"We show that this RNA cleavage step is essential for assembly of the Csy protein-crRNA complex that facilitates target recognition"
Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system.
-
The PA14 type I-F locus restricts replication of CRISPR-sensitive phages
"The CRISPR-sensitive phages fail to replicate on PA14 due to the action of the CRISPR/Cas system14, but are able to replicate on PA14"
Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins.
-
Anti-CRISPR proteins block the DNA-binding activity of the assembled type I-F complex by contacting different subunits
"Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits"
Foreign DNA acquisition by the I-F CRISPR-Cas system requires all components of the interference machinery.
-
Both naive and primed adaptation by the type I-F system require the intact Csy complex and crRNA
"both modes require, in addition to Cas1 and Cas2, intact Csy complex, an ortholog of the E. coli Cascade, and crRNA"
Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.
-
The structural work is on the Pseudomonas aeruginosa type I-F Csy complex
"The structural studies we describe here are focused on the type I-F Csy (CRISPR system yersinia) found in Pseudomonas aeruginosa"
-
Subunit positions on the crRNA, with Cas5f and Cas8f at the 5' handle
"Cas6f is located at the 3′ stem-loop of the crRNA, Cas8f and Cas5f are located at the 5′ handle of crRNA, and six interlocking copies of Cas7f are located along the length of the crRNA spacer"
-
The Cas5f thumb domain contributes to the pocket that clamps the PAM-containing target duplex
"the duplex region of the target DNA (including the G-C/G-C PAM) is sandwiched between the Cas8f N-terminal hook domain (residues 1-166), the Cas5f thumb domain (residues 48-109), and Cas7"
-
One Cas5f thumb residue lies in the DNA-binding pocket, which is dominated by Cas8f
"Numerous positively charged residues line this DNA binding pocket, including residues from Cas7.6f (K299), the Cas5f thumb (R90), and especially the Cas8f hook (R24, K28, K31, R59, K71 and R78)"
Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR-Cas complex revealed by cryo-EM.
-
Composition and stoichiometry of the type I-F Csy surveillance complex
"The Csy complex is comprised of four types of Cas proteins (Cas5f-8f) and a single 60-nt crRNA"
-
Cas5f and Cas8f occupy the tail, Cas6f the head, six Cas7f the backbone
"the maps exhibit an asymmetric spiral, with one Cas6f subunit in the head, one Cas5f and one Cas8f in the tail, and six Cas7f comprising the spiral backbone"
-
Cas5f and Cas8f clamp the crRNA 5' handle into an S shape
"the Cas5f and Cas8f subunits in the tail region also make extensive interactions with crRNA, causing the 8 nt of crRNA at its 5′ terminus to display an “S”-shape architecture, called the 5′ handle"
-
Recognition of the crRNA 5' handle by Cas5f and Cas8f is sequence-specific
"supporting a sequence-specific recognition of the crRNA 5′ handle by Cas5f and Cas8f"