New design strategies for ultra-specific CRISPR-Cas13a-based RNA-diagnostic tools with single-nucleotide mismatch sensitivity

Vargas, Adrian M. Molina; Osborn, Raven M; Sinha, Souvik; Arantes, Pablo Ricardo; Patel, Amun C.; Dewhurst, Stephen; Palermo, Giulia; O’Connell, Mitchell R.

doi:10.1101/2023.07.26.550755

Cited by 6 publications

(2 citation statements)

References 79 publications

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“…For example Quan et al (2019) used 5,513 sgRNAs to target 127 genes, and Ackerman et al (2020) used more than 4,500 sgRNAs in one assay to detect 169 different species with no detectable reduction in reaction efficiency. Additionally, CRISPR-Cas enrichment and depletion offer solutions to several challenges encountered in current eDNA monitoring methods, including the potential for quantitative assessment by avoiding PCR limitations, streamlining lengthy laboratory stages, and by increasing specificity/accuracy allowing for portable and isothermal assessment (Cai et al, 2024;Islam & Kasfy, 2023;Vargas et al, 2023).…”

Section: Enrichment Of Low-abundant Dnamentioning

confidence: 99%

Enhancing biodiversity monitoring efficiency through CRISPR-driven depletion and enrichment of aquatic environmental DNA

Kardailsky,

Durán-Vinet,

Nester

et al. 2024

Preprint

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Characterising biodiversity using environmental DNA (eDNA) represents a paradigm shift in our capacity for biomonitoring complex aquatic environments. However, eDNA biomonitoring is limited by biases towards certain species and low taxonomic resolution of current metabarcoding-based approaches. Shotgun metagenomics of eDNA enables the collection of whole ecosystem data by sequencing all molecules present in a sample, allowing them to be characterised and identified. Ongoing enhancements of whole genome reference databases are improving the resolution of shotgun metagenomics, reducing database related limitations in species and individual identification for metagenomic studies. However, shotgun metagenomics-based insights are constrained by preferential sequencing, favouring more abundant organisms in the water column like bacteria and viruses over less abundant target species like vertebrates. To improve the probability of detecting low abundant target organisms, methods such as target species enrichment or the removal of non-target DNA prior to sequencing can be employed. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated proteins (Cas) have recently emerged as a novel technology that can achieve both enrichment and depletion. CRISPR-Cas-based methods have the potential to improve the efficiency of eDNA metagenomic sequencing and simplify data analysis by reducing non-target data filtration steps, however, they have not been widely implemented due to a lack of interest and support in the past, as well as limited number of studies demonstrating they can be applied in a robust and cost-effective manner. Here, we review current approaches of CRISPR-Cas to study underrepresented aquatic taxa. We advocate that further optimization of depletion and enrichment methods of eDNA using CRISPR-Cas holds great promise for the rapidly evolving field of eDNA biomonitoring through refining monitoring approaches, overcoming PCR bias, and enabling efficient high-throughput applications.

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Section: Enrichment Of Low-abundant Dnamentioning

confidence: 99%

Enhancing biodiversity monitoring efficiency through CRISPR-driven depletion and enrichment of aquatic environmental DNA

Kardailsky,

Durán-Vinet,

Nester

et al. 2024

Preprint

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“…Models including only sequence effects while excluding structural effects have had success with a binary classification of active/inactive crRNAs, but have not been able to solve the regression problem of quantitatively predicting Cas13 activity from the crRNA sequence 10,11,14 . Second, although Cas13 activation requires crRNA/protospacer complementarity, Cas13 is frequently activated to a similar or even greater degree in the presence of single mismatches 15,16 . We hypothesized that these two poorly understood characteristics of Cas13 could be addressed by studying the activation of Cas13 in more detail using structured RNAs as a model system.…”

Section: Main Textmentioning

confidence: 99%

RNA structure modulates Cas13 activity and enables mismatch detection

Kimchi,

Larsen,

Dunkley

et al. 2023

Preprint

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The RNA-targeting CRISPR nuclease Cas13 has emerged as a powerful tool for applications ranging from nucleic acid detection to transcriptome engineering and RNA imaging. Cas13 is activated by the hybridization of a CRISPR RNA (crRNA) to a complementary single-stranded RNA (ssRNA) protospacer in a target RNA. Though Cas13 is not activated by double-stranded RNA (dsRNA)in vitro, it paradoxically demonstrates robust RNA targeting in environments where the vast majority of RNAs are highly structured. Understanding Cas13’s mechanism of binding and activation will be key to improving its ability to detect and perturb RNA; however, the mechanism by which Cas13 binds structured RNAs remains unknown. Here, we systematically probe the mechanism of LwaCas13a activation in response to RNA structure perturbations using a massively multiplexed screen. We find that there are two distinct sequence-independent modes by which secondary structure affects Cas13 activity: structure in the protospacer region competes with the crRNA and can be disrupted via a strand-displacement mechanism, while structure in the region 3’ to the protospacer has an allosteric inhibitory effect. We leverage the kinetic nature of the strand displacement process to improve Cas13-based RNA detection and enhance mismatch discrimination by up to 50-fold, enabling sequence-agnostic mutation identification at low (<1%) allele frequencies. Our work sets a new standard for CRISPR-based nucleic acid detection and will enable intelligent and secondary-structure-guided target selection while also expanding the range of RNAs available for targeting with Cas13.

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Tracing Allostery in the Spliceosome Ski2-like RNA Helicase Brr2

Guidarelli Mattioli,

Saltalamacchia,

Magistrato

2024

J. Phys. Chem. Lett.

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RNA ATPases/helicases remodel substrate RNA−protein complexes in distinct ways. The different RNA ATPases/ helicases, taking part in the spliceosome complex, reshape the RNA/RNA-protein contacts to enable premature-mRNA splicing. Among them, the bad response to refrigeration 2 (Brr2) helicase promotes U4/U6 small nuclear (sn)RNA unwinding via ATPdriven translocation of the U4 snRNA strand, thus playing a pivotal role during the activation, catalytic, and disassembly phases of splicing. The plastic Brr2 architecture consists of an enzymatically active N-terminal cassette (N-cassette) and a structurally similar but inactive C-terminal cassette (C-cassette). The C-cassette, along with other allosteric effectors and regulators, tightly and timely controls Brr2's function via an elusive mechanism. Here, microsecond-long molecular dynamics simulations, dynamical network theory, and community network analysis are combined to elucidate how allosteric effectors/regulators modulate the Brr2 function. We unexpectedly reveal that U4 snRNA itself acts as an allosteric regulator, amplifying the cross-talk of distal Brr2 domains and triggering a conformational reorganization of the protein. Our findings offer fundamental understanding into Brr2's mechanism of action and broaden our knowledge on the sophisticated regulatory mechanisms by which spliceosome ATPases/helicases control gene expression. This includes their allosteric regulation exerted by client RNA strands, a mechanism that may be broadly applicable to other RNA-dependent ATPases/helicases.

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New design strategies for ultra-specific CRISPR-Cas13a-based RNA-diagnostic tools with single-nucleotide mismatch sensitivity

Cited by 6 publications

References 79 publications

Enhancing biodiversity monitoring efficiency through CRISPR-driven depletion and enrichment of aquatic environmental DNA

Enhancing biodiversity monitoring efficiency through CRISPR-driven depletion and enrichment of aquatic environmental DNA

RNA structure modulates Cas13 activity and enables mismatch detection

Tracing Allostery in the Spliceosome Ski2-like RNA Helicase Brr2

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