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Distribution and molecular evolution of the anti-CRISPR family AcrIF7.

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Cazares, Daniel 
Wu, Yi 
de la Cruz, Ana 


Anti-clustered regularly interspaced short palindromic repeats (CRISPRs) are proteins capable of blocking CRISPR-Cas systems and typically their genes are located on mobile genetic elements. Since their discovery, numerous anti-CRISPR families have been identified. However, little is known about the distribution and sequence diversity of members within a family, nor how these traits influence the anti-CRISPR's function and evolution. Here, we use AcrIF7 to explore the dissemination and molecular evolution of an anti-CRISPR family. We uncovered 5 subclusters and prevalent anti-CRISPR variants within the group. Remarkably, AcrIF7 homologs display high similarity despite their broad geographical, ecological, and temporal distribution. Although mainly associated with Pseudomonas aeruginosa, AcrIF7 was identified in distinct genetic backgrounds indicating horizontal dissemination, primarily by phages. Using mutagenesis, we recreated variation observed in databases but also extended the sequence diversity of the group. Characterisation of the variants identified residues key for the anti-CRISPR function and other contributing to its mutational tolerance. Moreover, molecular docking revealed that variants with affected function lose key interactions with its CRISPR-Cas target. Analysis of publicly available data and the generated variants suggests that the dominant AcrIF7 variant corresponds to the minimal and optimal anti-CRISPR selected in the family. Our study provides a blueprint to investigate the molecular evolution of anti-CRISPR families.


Acknowledgements: We thank Guadalupe Aguilar González from Unidad de Ácidos Nucleicos, Department of Genetics and Molecular Biology and Dr. Dulce Delgadillo Álvarez from Unidad de Genómica, Proteómica y Metabolómica—CINVESTAV, for technical assistance in Sanger sequencing; and Miguel Ángel Moreno Galeana and Dr. Eva Jacinto from DGMB, CINVESTAV, for technical support with lab experiments. We also thank Dr. Herminia Loza Tavera from the Faculty of Chemistry, UNAM, for providing us with pUCP24 plasmid and Prof Alan Davidson for sharing with us the strains PA14 WT, PA14 ΔCR, and the phage JBD18.


Humans, Molecular Docking Simulation, CRISPR-Cas Systems, Bacteriophages, Evolution, Molecular, Mutation

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PLoS Biol

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Public Library of Science (PLoS)
Cambridge Trust (10469474)
Consejo Nacional de Ciencia y Tecnología (591274 and 706017)
European Molecular Biology Laboratory (EMBL-EBI/Wellcome Trust Sanger Institute Join Post-Doctoral Fellowship Program (ESPOD))
Consejo Nacional de Ciencia y Tecnología (586079)
Consejo Nacional de Ciencia y Tecnología (255255)