Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43
Abstract
Viruses within a given family often share common essential genes that are highly conserved due to their critical role for the virus’s replication and survival. In this work, we developed a proof-of-concept for a pan-coronavirus CRISPR effector system by designing CRISPR targets that are cross-reactive among essential genes of different human coronaviruses (HCoV). We designed CRISPR targets for both the RNA-dependent RNA polymerase (RdRp) gene as well as the nucleocapsid (N) gene in coronaviruses. Using sequencing alignment, we determined the most highly conserved regions of these genes to design guide RNA (gRNA) sequences. In regions that were not completely homologous among HCoV species, we introduced mismatches into the gRNA sequence and tested the efficacy of CasRx, a Cas13d type CRISPR effector, using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in HCoV-OC43. We evaluated the effect that mismatches in gRNA sequences has on the cleavage activity of CasRx and found that this CRISPR effector can tolerate up to three mismatches while still maintaining its nuclease activity in HCoV-OC43 viral RNA. This work highlights the need to evaluate off-target effects of CasRx with gRNAs containing up to three mismatches in order to design safe and effective CRISPR experiments.
- Authors:
-
[1];
- Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); University of Nebraska Medical Center, Omaha, NE (United States)
- University of Nebraska Medical Center, Omaha, NE (United States)
- Publication Date:
- Research Org.:
- Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
- Sponsoring Org.:
- USDOE National Nuclear Security Administration (NNSA)
- OSTI Identifier:
- 1877122
- Report Number(s):
- SAND2022-8295J
Journal ID: ISSN 2073-4409; 707443
- Grant/Contract Number:
- NA0003525
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Cells
- Additional Journal Information:
- Journal Volume: 11; Journal Issue: 12; Journal ID: ISSN 2073-4409
- Publisher:
- MDPI
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; CRISPR; CasRx; off-target effects; human coronaviruses; HCoV-OC43; homology
Citation Formats
Mayes, Cathryn Michelle, and Santarpia, Joshua. Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43. United States: N. p., 2022.
Web. doi:10.3390/cells11121859.
Mayes, Cathryn Michelle, & Santarpia, Joshua. Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43. United States. https://doi.org/10.3390/cells11121859
Mayes, Cathryn Michelle, and Santarpia, Joshua. Tue .
"Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43". United States. https://doi.org/10.3390/cells11121859. https://www.osti.gov/servlets/purl/1877122.
@article{osti_1877122,
title = {Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43},
author = {Mayes, Cathryn Michelle and Santarpia, Joshua},
abstractNote = {Viruses within a given family often share common essential genes that are highly conserved due to their critical role for the virus’s replication and survival. In this work, we developed a proof-of-concept for a pan-coronavirus CRISPR effector system by designing CRISPR targets that are cross-reactive among essential genes of different human coronaviruses (HCoV). We designed CRISPR targets for both the RNA-dependent RNA polymerase (RdRp) gene as well as the nucleocapsid (N) gene in coronaviruses. Using sequencing alignment, we determined the most highly conserved regions of these genes to design guide RNA (gRNA) sequences. In regions that were not completely homologous among HCoV species, we introduced mismatches into the gRNA sequence and tested the efficacy of CasRx, a Cas13d type CRISPR effector, using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in HCoV-OC43. We evaluated the effect that mismatches in gRNA sequences has on the cleavage activity of CasRx and found that this CRISPR effector can tolerate up to three mismatches while still maintaining its nuclease activity in HCoV-OC43 viral RNA. This work highlights the need to evaluate off-target effects of CasRx with gRNAs containing up to three mismatches in order to design safe and effective CRISPR experiments.},
doi = {10.3390/cells11121859},
journal = {Cells},
number = 12,
volume = 11,
place = {United States},
year = {Tue Jun 07 00:00:00 EDT 2022},
month = {Tue Jun 07 00:00:00 EDT 2022}
}
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