CRISPR-based engineering of RNA viruses

Nemudryi, Artem; Nemudraia, Anna; Nichols, Joseph E.; Scherffius, Andrew M.; Zahl, Trevor; Wiedenheft, Blake

doi:10.1126/sciadv.adj8277

Cited by 5 publications

(4 citation statements)

References 44 publications

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“…Precise DNA manipulation has been enabled by breaking DNA at sequence-specific locations and repairing the resulting fragments. In a previous study we applied the concept of break-and-repair to engineer viral RNA genomes in vitro ( 37 ). We extend this approach to make RNA deletions in human cells, which is facilitated by the repair of programmable RNA breaks made using type-III CRISPR complexes.…”

Section: Discussionmentioning

confidence: 99%

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

Nemudraia,

Nemudryi,

Wiedenheft

2024

Science

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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. Here, we show that site-specific RNA breaks generated with type III CRISPR complexes are repaired in human cells, and 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.

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Section: Discussionmentioning

confidence: 99%

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

Nemudraia,

Nemudryi,

Wiedenheft

2024

Science

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“…Precise DNA manipulation, in a test tube or in a cell, has been enabled by breaking DNA at sequence-specific locations and repairing the resulting fragments. In a previous study, we applied the same concept of break-and-repair to engineer viral RNA genomes in vitro (24). Here, we extend this approach to make RNA deletions in human cells, which is facilitated by the repair of programmable RNA breaks made using type III CRISPR complexes.…”

Section: Discussionmentioning

confidence: 99%

Repair of CRISPR-guided RNA breaks enables site-specific RNA editing in human cells

Nemudraia,

Nemudryi,

Wiedenheft

2023

Preprint

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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. Here, we show that site-specific RNA breaks generated with RNA-targeting CRISPR complexes are repaired in human cells, and this repair can be used for programmable deletions in human transcripts that restore gene function. Collectively, this work establishes a technology for precise RNA manipulation with potential therapeutic applications.One-Sentence SummaryCRISPR-guided RNA breaks are repaired in human cells, and this RNA repair can be used for programmable editing of human transcriptomes.

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“…A similar recombination phenomenon can occur during dsRNA phage replication (step 3 ), after which genome reassortment can lead to various combinations of packaged genomic segments (step 4 ) [127]. (3) Programmable RNA editing using guide RNA (crRNA) and CRISPRguided nuclease (CRISPR-Cms) cleavage followed by RNA ligase-mediated strand repair in the presence of a synthetic DNA splint (adapted from [128]). ( 4) Synthetic platform for the production of viral VLPs from metaviromic data.…”

Section: Techniques For Genetic Engineering Of Ssdna and Rna Phagesmentioning

confidence: 99%

“…In September 2023, Nemudryi and colleagues unveiled an innovative CRISPR/Casbased RNA editing technique facilitating rapid and programmable deletions, insertions, and substitutions in RNA without the need for DNA intermediates [128]. By combining type III CRISPR/Cas-based RNA cleavage with splinted RNA ligation, the authors achieved targeted modifications in RNA sequences.…”

Section: Techniques For Genetic Engineering Of Ssdna and Rna Phagesmentioning

confidence: 99%

RNA and Single-Stranded DNA Phages: Unveiling the Promise from the Underexplored World of Viruses

Nguyen,

Watanabe,

Sharmin

et al. 2023

IJMS

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RNA and single-stranded DNA (ssDNA) phages make up an understudied subset of bacteriophages that have been rapidly expanding in the last decade thanks to advancements in metaviromics. Since their discovery, applications of genetic engineering to ssDNA and RNA phages have revealed their immense potential for diverse applications in healthcare and biotechnology. In this review, we explore the past and present applications of this underexplored group of phages, particularly their current usage as therapeutic agents against multidrug-resistant bacteria. We also discuss engineering techniques such as recombinant expression, CRISPR/Cas-based genome editing, and synthetic rebooting of phage-like particles for their role in tailoring phages for disease treatment, imaging, biomaterial development, and delivery systems. Recent breakthroughs in RNA phage engineering techniques are especially highlighted. We conclude with a perspective on challenges and future prospects, emphasizing the untapped diversity of ssDNA and RNA phages and their potential to revolutionize biotechnology and medicine.

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CRISPR-based engineering of RNA viruses

Cited by 5 publications

References 44 publications

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

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

Repair of CRISPR-guided RNA breaks enables site-specific RNA editing in human cells

RNA and Single-Stranded DNA Phages: Unveiling the Promise from the Underexplored World of Viruses

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