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Identification of PAM Requirements for the Vibrio cholerae type I-E CRISPR-Cas System
Anne M Stringer1, Joseph T Wade1,2,3
1Wadsworth Center, New York State Department of Health, Albany, New York, USA.
Abstract:
CRISPR-Cas systems are prokaryotic adaptive immune systems that use RNA-guided protein complexes to target invading nucleic acid. A surveillance complex consisting of protein and a CRISPR-RNA (crRNA) binds target nucleic acid via base-pairing interactions, typically leading to processing of the target nucleic acid by a nuclease. CRISPR-Cas systems are classified based on their mechanism of action, with type I systems being the most prevalent in nature. Type I CRISPR-Cas systems target DNA, and require extensive complementarity between the crRNA and the target DNA. Moreover, type I systems require the presence of a "Protospacer Adjacent Motif" (PAM) sequence in the target DNA immediately adjacent to the expected region of base-pairing with the crRNA. Classical biotypes of the bacterial pathogen Vibrio cholerae have active type I-E CRISPR-Cas systems. While the optimal PAM sequence for this CRISPR-Cas system is known to be AAY, the activity of other sequences as possible PAMs has not been determined. Here, we quantify the effectiveness of all possible trinucleotide sequences in the PAM position for the V. cholerae type I-E CRISPR-Cas system. Our data indicate a hierarchy of PAM efficacy, with 15 of the 64 trinucleotide sequences functioning as a PAM.
Insights
Researchers investigated the Protospacer Adjacent Motif (PAM) sequences for the Vibrio cholerae type I-E CRISPR-Cas system. They found 15 out of 64 possible trinucleotide sequences effectively function as a PAM, revealing a hierarchy of efficacy.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- CRISPR-Cas systems provide prokaryotes with adaptive immunity against foreign nucleic acids.
- Type I CRISPR-Cas systems, prevalent in nature, target DNA using RNA-guided nucleases and require a Protospacer Adjacent Motif (PAM) for target recognition.
- The bacterial pathogen Vibrio cholerae possesses active type I-E CRISPR-Cas systems, with a known optimal PAM sequence of AAY.
Purpose of the Study:
- To systematically evaluate the efficacy of all possible trinucleotide sequences as PAMs for the Vibrio cholerae type I-E CRISPR-Cas system.
- To establish a hierarchy of PAM efficacy for this specific CRISPR-Cas subtype.
Main Methods:
- Quantification of the effectiveness of all 64 possible trinucleotide sequences at the PAM position.
- Utilizing the Vibrio cholerae type I-E CRISPR-Cas system as the model.
Main Results:
- A defined hierarchy of PAM efficacy was identified for the V. cholerae type I-E CRISPR-Cas system.
- Fifteen out of the 64 tested trinucleotide sequences demonstrated functionality as a PAM.
Conclusions:
- The study expands the known PAM recognition capabilities of the V. cholerae type I-E CRISPR-Cas system beyond the optimal AAY sequence.
- Understanding PAM sequence hierarchy is crucial for optimizing CRISPR-Cas applications and studying its natural function.
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