CRISPR-Cas12a target binding unleashes single-stranded DNase activity, bioRxiv, 2017-11-30

AbstractCRISPR-Cas12a (Cpf1) proteins are RNA-guided DNA targeting enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a can be used as a powerful genome editing tool based on its ability to induce genetic changes in cells at sites of double-stranded DNA (dsDNA) cuts. Here we show that RNA-guided DNA binding unleashes robust, non-specific single-stranded DNA (ssDNA) cleavage activity in Cas12a sufficient to completely degrade both linear and circular ssDNA molecules within minutes. This activity, catalyzed by the same active site responsible for site-specific dsDNA cutting, indiscriminately shreds ssDNA with rapid multiple-turnover cleavage kinetics. Activation of ssDNA cutting requires faithful recognition of a DNA target sequence matching the 20-nucleotide guide RNA sequence with specificity sufficient to distinguish between closely related viral serotypes. We find that target-dependent ssDNA degradation, not observed for CRISPR-Cas9 enzymes, is a fundamental property of type V CRISPR-Cas12 proteins, revealing a fascinating parallel with the RNA-triggered general RNase activity of the type VI CRISPR-Cas13 enzymes.One Sentence SummaryCas12a (Cpf1) and related type V CRISPR interference proteins possess non-specific, single-stranded DNase activity upon activation by guide RNA-dependent DNA binding.

biorxiv biochemistry 0-100-users 2017

High-throughput ANI Analysis of 90K Prokaryotic Genomes Reveals Clear Species Boundaries, bioRxiv, 2017-11-28

A fundamental question in microbiology is whether there is a continuum of genetic diversity among genomes or clear species boundaries prevail instead. Answering this question requires robust measurement of whole-genome relatedness among thousands of genomes and from diverge phylogenetic lineages. Whole-genome similarity metrics such as Average Nucleotide Identity (ANI) can provide the resolution needed for this task, overcoming several limitations of traditional techniques used for the same purposes. Although the number of genomes currently available may be adequate, the associated bioinformatics tools for analysis are lagging behind these developments and cannot scale to large datasets. Here, we present a new method, FastANI, to compute ANI using alignment-free approximate sequence mapping. Our analyses demonstrate that FastANI produces an accurate ANI estimate and is up to three orders of magnitude faster when compared to an alignment (e.g., BLAST)-based approach. We leverage FastANI to compute pairwise ANI values among all prokaryotic genomes available in the NCBI database. Our results reveal a clear genetic discontinuity among the database genomes, with 99.8% of the total 8 billion genome pairs analyzed showing either >95% intra-species ANI or <83% inter-species ANI values. We further show that this discontinuity is recovered with or without the most frequently represented species in the database and is robust to historic additions in the public genome databases. Therefore, 95% ANI represents an accurate threshold for demarcating almost all currently named prokaryotic species, and wide species boundaries may exist for prokaryotes.

biorxiv bioinformatics 200-500-users 2017

 

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