Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium
Name
23100.full.pdf
Description
Published version
Size
1.05 MB
Format
Adobe PDF
Checksum (MD5)
c4571ecfc93947ac8ebdd19d21a56871
Author(s)
Blainey, Paul C
Date Issued
October 28, 2019
Journal
Proceedings of the National Academy of Sciences of the United States of America
Publisher
Proceedings of the National Academy of Sciences
Citation
Schmidt, Stephanie Tzouanas et al. "Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium." Proceedings of the National Academy of Sciences of the United States of America 116(2019):23100-23105 © 2019 The Author(s)
Version
Final published version
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) systems have been effectively harnessed to engineer the genomes of organisms from across the tree of life. Nearly all currently characterized Cas9 proteins are derived from mesophilic bacteria, and canonical Cas9 systems are challenged by applications requiring enhanced stability or elevated temperatures. We discovered IgnaviCas9, a Cas9 protein from a hyperthermophilic Ignavibacterium identified through mini-metagenomic sequencing of samples from a hot spring. IgnaviCas9 is active at temperatures up to 100 °C in vitro, which enables DNA cleavage beyond the 44 °C limit of Streptococcus pyogenes Cas9 (SpyCas9) and the 70 °C limit of both Geobacillus stearothermophilus Cas9 (GeoCas9) and Geobacillus thermodenitrificans T12 Cas9 (ThermoCas9). As a potential application of this enzyme, we demonstrate that IgnaviCas9 can be used in bacterial RNA-seq library preparation to remove unwanted cDNA from 16s ribosomal rRNA without increasing the number of steps, thus underscoring the benefits provided by its exceptional thermostability in improving molecular biology and genomic workflows. IgnaviCas9 is an exciting addition to the CRISPR-Cas9 toolbox and expands its temperature range.
Subjects
Multidisciplinary
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Biological Engineering
Terms of Use
Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.1073/pnas.1904273116
