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

Nemudraia, Anna; Nemudryi, Artem; Wiedenheft, Blake

doi:10.1126/science.adk5518

Cited by 3 publications

References 64 publications

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CRISPR-Cas target recognition for sensing viral and cancer biomarkers

Rahimi,

Balusamy,

Perumalsamy

et al. 2024

Nucleic Acids Research

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Nucleic acid-based diagnostics is a promising venue for detection of pathogens causing infectious diseases and mutations related to cancer. However, this type of diagnostics still faces certain challenges, and there is a need for more robust, simple and cost-effective methods. Clustered regularly interspaced short palindromic repeats (CRISPRs), the adaptive immune systems present in the prokaryotes, has recently been developed for specific detection of nucleic acids. In this review, structural and functional differences of CRISPR-Cas proteins Cas9, Cas12 and Cas13 are outlined. Thereafter, recent reports about applications of these Cas proteins for detection of viral genomes and cancer biomarkers are discussed. Further, we highlight the challenges associated with using these technologies to replace the current diagnostic approaches and outline the points that need to be considered for designing an ideal Cas-based detection system for nucleic acids.

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CRISPR-Cas target recognition for sensing viral and cancer biomarkers

Rahimi,

Balusamy,

Perumalsamy

et al. 2024

Nucleic Acids Research

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Single-molecule live-cell RNA imaging with CRISPR-Csm

Xia,

Colognori,

Jiang

et al. 2024

Preprint

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High-resolution, real-time imaging of RNA is essential for understanding the diverse, dynamic behaviors of individual RNA molecules in single cells. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved. Here, we present single-molecule live-cell fluorescencein situhybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR-Csm complex with multiplexed guide RNAs for efficient, direct visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we tracked individual endogenousNOTCH2andMAP1BmRNA transcripts in living cells and identified two distinct localization mechanisms: co-translational translocation ofNOTCH2mRNA at the endoplasmic reticulum, and directional transport ofMAP1BmRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.

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CRISPR-RNA binding drives structural ordering that primes Cas7-11 for target cleavage

Lin,

Li,

Brogan

et al. 2024

Preprint

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Type III-E CRISPR-Cas effectors, of which Cas7-11 is the first, are single proteins that cleave target RNAs without nonspecific collateral cleavage, opening new possibilities for RNA editing. Biochemical experiments combined with amide hydrogen-deuterium exchange (HDX-MS) experiments provide a first glimpse of the conformational dynamics of apo Cas7-11. HDX-MS revealed the backbone comprised of the four Cas7 zinc-binding RRM folds are well-folded but insertion sequences are highly dynamic and fold upon binding crRNA. The crRNA causes folding of disordered catalytic loops and β-hairpins, stronger interactions at domain-domain interfaces, and folding of the Cas7.1 processing site. Target RNA binding causes only minor ordering around the catalytic loops of Cas7.2 and Cas7.3. We show that Cas7-11 cannot fully process the CRISPR array and that binding of partially processed crRNA induces multiple states in Cas7-11 and reduces target RNA cleavage. The insertion domain shows the most ordering upon binding of mature crRNA. Finally, we show a crRNA-induced conformational change in one of the TPR-CHAT binding sites providing an explanation for why crRNA binding facilitates TPR-CHAT binding. The results provide the first glimpse of the apo state of Cas7-11 and reveal how its structure and function are regulated by crRNA binding.

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

Cited by 3 publications

References 64 publications

CRISPR-Cas target recognition for sensing viral and cancer biomarkers

CRISPR-Cas target recognition for sensing viral and cancer biomarkers

Single-molecule live-cell RNA imaging with CRISPR-Csm

CRISPR-RNA binding drives structural ordering that primes Cas7-11 for target cleavage

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