Transcriptome-wide Cas13 guide RNA design for model organisms and viral RNA pathogens

Guo, Xinyi; Rahman, Jahan; Wessels, Hans-Hermann; Méndez-Mancilla, Alejandro; Haro, Daniel; Chen, Xinru; Sanjana, Neville E.

doi:10.1016/j.xgen.2021.100001

Cited by 37 publications

(35 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To account for this, we introduced up to three mismatches in the gRNA sequences to determine how effective the CasRx endonuclease is when there is sequence variability between the gRNA and target sequences. Previous work has analyzed the effect that gRNA mismatches have on Cas13 efficacy both computationally [ 6 ] and experimentally in organisms such as Drosophila [ 7 ] and human and mouse cells [ 8 ]. This work has found that Cas13 effectors may be tolerant to certain nucleotide mismatches [ 9 , 10 ], especially when present in the central region of the gRNA [ 6 , 7 ].…”

Section: Introductionmentioning

confidence: 99%

Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43

Mayes

Santarpia

2022

Cells

View full text Add to dashboard Cite

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.

show abstract

Section: Introductionmentioning

confidence: 99%

Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43

Mayes

Santarpia

2022

Cells

View full text Add to dashboard Cite

show abstract

“…Several bioinformatics tools aim to help select spacer sequences for Cas9 systems targeting DNA [ 65 ]. Tools for non-Cas9 systems have also been developed and often consider the secondary structure of both the gRNA and target RNA [ 66 , 67 , 68 ]. For example, Wessels et al created an online tool that predicts Cas13d gRNAs for all protein-coding transcripts in the human genome (cas13design.nygenome.org) [ 62 ].…”

Section: Step-by-step Selection and Design Of Rna-targeting Crispr-ca...mentioning

confidence: 99%

How to Find the Right RNA-Sensing CRISPR-Cas System for an In Vitro Application

2022

View full text Add to dashboard Cite

CRISPR-Cas systems have a great and still largely untapped potential for in vitro applications, in particular, for RNA biosensing. However, there is currently no systematic guide on selecting the most appropriate RNA-targeting CRISPR-Cas system for a given application among thousands of potential candidates. We provide an overview of the currently described Cas effector systems and review existing Cas-based RNA detection methods. We then propose a set of systematic selection criteria for selecting CRISPR-Cas candidates for new applications. Using this approach, we identify four candidates for in vitro RNA.

show abstract

“…In this context, Cas 13 is an endonuclease that targets and binds sg RNA. Moreover, it demonstrates RNA cis-cleavage activity when activated by a target RNA in different model organisms [165] and is more effective and specific than RNAi in mammalian cells [166,167]. For this purpose, CRISPR-Cas13 has been proposed and used as a tool for SARS-CoV-2 detection [168].…”

Section: Gene Editingmentioning

confidence: 99%

mRNA, a Revolution in Biomedicine

et al. 2021

View full text Add to dashboard Cite

The perspective of using messenger RNA (mRNA) as a therapeutic molecule first faced some uncertainties due to concerns about its instability and the feasibility of large-scale production. Today, given technological advances and deeper biomolecular knowledge, these issues have started to be addressed and some strategies are being exploited to overcome the limitations. Thus, the potential of mRNA has become increasingly recognized for the development of new innovative therapeutics, envisioning its application in immunotherapy, regenerative medicine, vaccination, and gene editing. Nonetheless, to fully potentiate mRNA therapeutic application, its efficient production, stabilization and delivery into the target cells are required. In recent years, intensive research has been carried out in this field in order to bring new and effective solutions towards the stabilization and delivery of mRNA. Presently, the therapeutic potential of mRNA is undoubtedly recognized, which was greatly reinforced by the results achieved in the battle against the COVID-19 pandemic, but there are still some issues that need to be improved, which are critically discussed in this review.

show abstract

Transcriptome-wide Cas13 guide RNA design for model organisms and viral RNA pathogens

Cited by 37 publications

References 32 publications

Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43

Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43

How to Find the Right RNA-Sensing CRISPR-Cas System for an In Vitro Application

mRNA, a Revolution in Biomedicine

Contact Info

Product

Resources

About