Cas4-Cas1 fusions drive efficient PAM selection and control CRISPR adaptation
C. Almendros Romero (Kavli institute of nanoscience Delft, TU Delft - BN/Stan Brouns Lab)
Franklin L. Luzia de Nobrega (TU Delft - BN/Stan Brouns Lab, Kavli institute of nanoscience Delft)
Rebecca E. McKenzie (TU Delft - BN/Stan Brouns Lab, Kavli institute of nanoscience Delft)
Stan J J Brouns (TU Delft - BN/Stan Brouns Lab, Kavli institute of nanoscience Delft, Wageningen University & Research)
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Abstract
Microbes have the unique ability to acquire immunological memories from mobile genetic invaders to protect themselves from predation. To confer CRISPR resistance, new spacers need to be compatible with a targeting requirement in the invader's DNA called the protospacer adjacent motif (PAM). Many CRISPR systems encode Cas4 proteins to ensure new spacers are integrated that meet this targeting prerequisite. Here we report that a gene fusion between cas4 and cas1 from the Geobacter sulfurreducens I-U CRISPR-Cas system is capable of introducing functional spacers carrying interference proficient TTN PAM sequences at much higher frequencies than unfused Cas4 adaptation modules. Mutations of Cas4-domain catalytic residues resulted in dramatically decreased naïve and primed spacer acquisition, and a loss of PAM selectivity showing that the Cas4 domain controls Cas1 activity. We propose the fusion gene evolved to drive the acquisition of only PAM-compatible spacers to optimize CRISPR interference.
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