title: Haloarcula hispanica CRISPR authenticates PAM of a target sequence to prime discriminative adaptation.
abstract: The prokaryotic immune system CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) adapts to foreign invaders by acquiring their short deoxyribonucleic acid (DNA) fragments as spacers, which guide subsequent interference to foreign nucleic acids based on sequence matching. The adaptation mechanism avoiding acquiring 'self' DNA fragments is poorly understood. In Haloarcula hispanica, we previously showed that CRISPR adaptation requires being primed by a pre-existing spacer partially matching the invader DNA. Here, we further demonstrate that flanking a fully-matched target sequence, a functional PAM (protospacer adjacent motif) is still required to prime adaptation. Interestingly, interference utilizes only four PAM sequences, whereas adaptation-priming tolerates as many as 23 PAM sequences. This relaxed PAM selectivity explains how adaptation-priming maximizes its tolerance of PAM mutations (that escape interference) while avoiding mis-targeting the spacer DNA within CRISPR locus. We propose that the primed adaptation, which hitches and cooperates with the interference pathway, distinguishes target from non-target by CRISPR ribonucleic acid guidance and PAM recognition.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Li
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China xiangh@im.ac.cn.
authors.forename: Hua
authors.initials: H
authors.lastname: Xiang
mesh_terms: D000220:Adaptation, Biological; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000737:chemistry; D019613:Haloarcula / Q000235:genetics*; D059372:Nucleotide Motifs; D009711:Nucleotides / Q000032:analysis
chemical_list: D009711:Nucleotides; D004247:DNA