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3 results on '"Nemudraia A"'

1. Repair of CRISPR-guided RNA breaks enables site-specific RNA excision in human cells.

Author

Nemudraia A, Nemudryi A, and Wiedenheft B

Subjects
Humans, DNA Repair, Endonucleases metabolism, HEK293 Cells, Protein Deglycase DJ-1 genetics, Cyclophilins genetics, Streptococcus thermophilus, CRISPR-Cas Systems, Gene Editing methods, RNA genetics, RNA, Guide, CRISPR-Cas Systems genetics, CRISPR-Associated Proteins
Abstract

Genome editing with CRISPR RNA-guided endonucleases generates DNA breaks that are resolved by cellular DNA repair machinery. However, analogous methods to manipulate RNA remain unavailable. We show that site-specific RNA breaks generated with type-III CRISPR complexes are repaired in human cells and that this repair can be used for programmable deletions in human transcripts to restore gene function. Collectively, this work establishes a technology for precise RNA manipulation with potential therapeutic applications.

Published
2024
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2. Intrinsic Signal Amplification by Type-III CRISPR-Cas Systems Provides a Sequence-Specific Viral Diagnostic

Author

Jodi F. Hedges, Laina N. Hall, Blake Wiedenheft, Deann T. Snyder, Tanner Wiegand, Anna Nemudraia, Pushya Krishna, Andrew Santiago-Frangos, Artem Nemudryi, Royce A. Wilkinson, Matthew P. Taylor, and Mark A. Jutila

Subjects
biology, Chemistry, Oligonucleotide, Specific detection, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), biology.protein, CRISPR, RNA, Computational biology, Signal amplification, Polymerase, Sequence (medicine)
Abstract

To combat viral pandemics, there is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here we repurposed the type III CRISPR-Cas system for sensitive and sequence specific detection of SARS-CoV-2 in an assay that can be performed in one hour or less. RNA recognition by type III systems triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons and cyclic oligonucleotides. We show that amplified products of the Cas10-polymerase are detectable using colorimetric or fluorometric readouts.

Published
2020

3. Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic

Author

Anna Nemudraia, Mark A. Jutila, Pushya Krishna, Deann T. Snyder, Laina N. Hall, Calvin Cicha, Jodi F. Hedges, Matthew P. Taylor, Tanner Wiegand, Artem Nemudryi, Royce A. Wilkinson, Blake Wiedenheft, Helen H Lee, Ava Graham, and Andrew Santiago-Frangos

Subjects
Medicine (General), type III, viral diagnostics, Computational biology, Genome, General Biochemistry, Genetics and Molecular Biology, R5-920, Report, Nasopharynx, Humans, CRISPR, Guide RNA, CRISPR-Cas, Polymerase, Sequence (medicine), Detection limit, CRISPR Dx, biology, SARS-CoV-2, Chemistry, Oligonucleotide, COVID-19, RNA, Molecular Diagnostic Techniques, COVID-19 Nucleic Acid Testing, biology.protein, RNA, Viral, Colorimetry, CRISPR-Cas Systems, point-of-care diagnostics, Nucleic Acid Amplification Techniques, RNA, Guide, Kinetoplastida
Abstract

There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/μL for a single guide RNA to 106 copies/μL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/μL and is completed using patient samples in less than 30 min., Graphical abstract, Recognition of a complementary target RNA by the type III CRISPR systems uniquely triggers the activation of a CRISPR-associated polymerase domain in Cas10. The polymerase generates oligoadenylates, protons, and pyrophosphates. Santiago-Frangos et al. repurposed the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2 by developing three different detection methods that rely on each of these products.

Published
2021

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