Two distinct DNA binding modes guide dual roles of a CRISPR-Cas protein complex.
Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets.
Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, 2628 CJ, Delft, The Netherlands.
Timothy R
TR
Blosser
Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, 2628 CJ, Delft, The Netherlands.
Luuk
L
Loeff
Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB, Wageningen, The Netherlands.
Edze R
ER
Westra
Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB, Wageningen, The Netherlands.
Marnix
M
Vlot
Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB, Wageningen, The Netherlands.
Tim
T
Künne
Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB, Wageningen, The Netherlands.
Małgorzata
M
Sobota
Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, 2628 CJ, Delft, The Netherlands.
Cees
C
Dekker
Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB, Wageningen, The Netherlands.
Stan J J
SJJ
Brouns
Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, 2628 CJ, Delft, The Netherlands.
Chirlmin
C
Joo
D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000276:immunology*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000276:immunology*; D003090:Coliphages / Q000737:chemistry / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology / Q000821:virology; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D015964:Gene Expression Regulation, Bacterial / Q000276:immunology*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding
D064130:CRISPR-Associated Proteins; D004279:DNA, Viral; D029968:Escherichia coli Proteins