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Distribution and phasing of sequence motifs that facilitate CRISPR adaptation.

ABSTRACT: CRISPR-associated proteins (Cas1 and Cas2) integrate foreign DNA at the "leader" end of CRISPR loci. Several CRISPR leader sequences are reported to contain a binding site for a DNA-bending protein called integration host factor (IHF). IHF-induced DNA bending kinks the leader of type I-E CRISPRs, recruiting an upstream sequence motif that helps dock Cas1-2 onto the first repeat of the CRISPR locus. To determine the prevalence of IHF-directed CRISPR adaptation, we analyzed 15,274 bacterial and archaeal CRISPR leaders. These experiments reveal multiple IHF binding sites and diverse upstream sequence motifs in a subset of the I-C, I-E, I-F, and II-C CRISPR leaders. We identify subtype-specific motifs and show that the phase of these motifs is critical for CRISPR adaptation. Collectively, this work clarifies the prevalence and mechanism(s) of IHF-dependent CRISPR adaptation and suggests that leader sequences and adaptation proteins may coevolve under the selective pressures of foreign genetic elements like plasmids or phages.

SUBMITTER: Santiago-Frangos A

PROVIDER: S-EPMC8552246 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Distribution and phasing of sequence motifs that facilitate CRISPR adaptation.

Santiago-Frangos Andrew A Buyukyoruk Murat M Wiegand Tanner T Krishna Pushya P Wiedenheft Blake B

Current biology : CB 20210625 16

CRISPR-associated proteins (Cas1 and Cas2) integrate foreign DNA at the "leader" end of CRISPR loci. Several CRISPR leader sequences are reported to contain a binding site for a DNA-bending protein called integration host factor (IHF). IHF-induced DNA bending kinks the leader of type I-E CRISPRs, recruiting an upstream sequence motif that helps dock Cas1-2 onto the first repeat of the CRISPR locus. To determine the prevalence of IHF-directed CRISPR adaptation, we analyzed 15,274 bacterial and ar ...[more]

PMID: 34174210

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Project description:Cation and anion channelrhodopsins (CCRs and ACRs, respectively) primarily from two algal species, Chlamydomonas reinhardtii and Guillardia theta, have become widely used as optogenetic tools to control cell membrane potential with light. We mined algal and other protist polynucleotide sequencing projects and metagenomic samples to identify 75 channelrhodopsin homologs from four channelrhodopsin families, including one revealed in dinoflagellates in this study. We carried out electrophysiological analysis of 33 natural channelrhodopsin variants from different phylogenetic lineages and 10 metagenomic homologs in search of sequence determinants of ion selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins. Our results show that association of a reduced number of glutamates near the conductance path with anion selectivity depends on a wider protein context, because prasinophyte homologs with a glutamate pattern identical to that in cryptophyte ACRs are cation selective. Desensitization is also broadly context dependent, as in one branch of stramenopile ACRs and their metagenomic homologs, its extent roughly correlates with phylogenetic relationship of their sequences. Regarding spectral tuning, we identified two prasinophyte CCRs with red-shifted spectra to 585 nm. They exhibit a third residue pattern in their retinal-binding pockets distinctly different from those of the only two types of red-shifted channelrhodopsins known (i.e., the CCR Chrimson and RubyACRs). In cryptophyte ACRs we identified three specific residue positions in the retinal-binding pocket that define the wavelength of their spectral maxima. Lastly, we found that dinoflagellate rhodopsins with a TCP motif in the third transmembrane helix and a metagenomic homolog exhibit channel activity. IMPORTANCE Channelrhodopsins are widely used in neuroscience and cardiology as research tools and are considered prospective therapeutics, but their natural diversity and mechanisms remain poorly characterized. Genomic and metagenomic sequencing projects are producing an ever-increasing wealth of data, whereas biophysical characterization of the encoded proteins lags behind. In this study, we used manual and automated patch clamp recording of representative members of four channelrhodopsin families, including a family in dinoflagellates that we report in this study. Our results contribute to a better understanding of molecular determinants of ionic selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins.

Dataset Information

Distribution and phasing of sequence motifs that facilitate CRISPR adaptation.

Publications

Distribution and phasing of sequence motifs that facilitate CRISPR adaptation.

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