Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43.
Cells
; 11(12)2022 06 07.
Article
in English
| MEDLINE | ID: covidwho-1884014
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.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Coronavirus Infections
/
Coronavirus
/
Coronavirus OC43, Human
Type of study:
Experimental Studies
/
Prognostic study
/
Randomized controlled trials
Limits:
Humans
Language:
English
Year:
2022
Document Type:
Article
Affiliation country:
Cells11121859
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