Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9

Autor:	Chunlei Jiao, Sahil Sharma, Lars Barquist, Yanying Yu, Oliver Kurzai, Christoph Schoen, Gaurav Dugar, Natalia L. Peeck, Franziska Wimmer, Cynthia M. Sharma, Thorsten Bischler, Chase L. Beisel
Rok vydání:	2021
Předmět:	Molec Biol Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Computational biology Campylobacter jejuni Bacterial genetics Nucleic acid thermodynamics CRISPR-Associated Protein 9 Humans CRISPR Clustered Regularly Interspaced Short Palindromic Repeats RNA Messenger Research Articles Gene Editing Trans-activating crRNA Multidisciplinary Base Sequence biology Diagnostic Tests Routine SARS-CoV-2 Cas9 R-Articles COVID-19 Nucleic Acid Hybridization RNA Microbio biology.organism_classification RNA Bacterial COVID-19 Nucleic Acid Testing Spike Glycoprotein Coronavirus RNA Viral CRISPR-Cas Systems Research Article RNA Guide Kinetoplastida
Zdroj:	Science (New York, N.y.)
ISSN:	1095-9203 0036-8075
Popis:	Cellular RNAs guide CRISPR-Cas9 The Cas9 nuclease widely used for genome editing is derived from natural bacterial defense systems that protect against invading viruses. Cas9 is directed by RNA guides to cut matching viral DNA. Jiao et al. discovered that RNA guides can also originate from cellular RNAs unassociated with viral defense (see the Perspective by Abudayyeh and Gootenberg). They rendered this process programmable, linking the presence of virtually any RNA to cutting of matching DNA by Cas9. This capability is the basis of a new CRISPR diagnostic method developed by the authors that can detect many biomarkers at once. Named LEOPARD, this method can detect, for example, RNAs from severe acute respiratory syndrome coronavirus 2 and other viruses, thereby translating a new CRISPR discovery into a powerful diagnostic tool. Science, abe7106, this issue p. 941; see also abi9335, p. 914 Cas9 guide RNAs can be derived from cellular RNAs, inspiring a multiplexable diagnostic platform. CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of “noncanonical” crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA of interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its D614G (Asp614→Gly) variant with single-base resolution in patient samples.
Databáze:	OpenAIRE
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