Recent developments and future directions in point-of-care next-generation CRISPR-based rapid diagnosis

Clinical and Experimental Medicine, Год журнала: 2025, Номер 25(1)

Опубликована: Янв. 9, 2025

Язык: Английский

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Comparison of CRISPR-Cas13b RNA base editing approaches for USH2A-associated inherited retinal degeneration

Communications Biology, Год журнала: 2025, Номер 8(1)

Опубликована: Фев. 8, 2025

CRISPR-Cas13 systems have therapeutic promise for the precise correction of point mutations in RNA. Using adenosine deaminase acting on RNA (ADAR) effectors, A-I base conversions can be targeted using guide RNAs (gRNAs). We compare Cas13 effectors PspCas13b and Cas13bt3 repair gene USH2A, a common cause inherited retinal disease Usher syndrome. In cultured cells, we demonstrate up to 80% efficiency c.11864 G > A its murine equivalent c.11840 A, across different gRNAs promoters. develop characterize mouse model syndrome carrying mutation designed evaluation editors disease. Finally, delivered via AAV Ush2a photoreceptors. Mean editing rates photoreceptors constructs ranged from 0.32% 2.04%, with greater those injected compared constructs. mice constructs, usherin protein was successfully restored correctly localized connecting cilium following editing. These results support development transcriptome targeting therapies

Язык: Английский

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Accurate Prediction of CRISPR/Cas13a Guide Activity Using Feature Selection and Deep Learning

Journal of Chemical Information and Modeling, Год журнала: 2025, Номер unknown

Опубликована: Март 17, 2025

CRISPR/Cas13a serves as a key tool for nucleic acid tests; therefore, accurate prediction of its activity is essential creating robust and sensitive diagnosis. In this study, we create dual-branch neural network model that achieves high accuracy classification performance across two independent data sets, outperforming previously published models relying solely on sequence features. The integrates direct encoding with descriptive features yields 99 out 1553, extracted through statistical analysis, which critically influence guide–target interactions Cas13a guide activity. By employing Shapley Additive Explanations Integrated Gradients feature importance show composition, mismatch type frequency, the protospacer flanking site region are primary These findings underscore using complementary inputs to deep learning-based provide valuable insights into mechanisms underlying interaction. All in all, study not only introduces reliable efficient but also offers foundation future rational design efforts.

Язык: Английский

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Systemic evaluation of various CRISPR/Cas13 orthologs for knockdown of targeted transcripts in plants

Genome biology, Год журнала: 2024, Номер 25(1)

Опубликована: Дек. 5, 2024

CRISPR/Cas13 system, recognized for its compact size and specificity in targeting RNA, is currently employed RNA degradation. However, the potential of various subtypes, particularly concerning knockdown endogenous transcripts, remains to be comprehensively characterized plants. Here we present a full spectrum editing profiles seven Cas13 orthologs from five distinct subtypes: VI-A (LwaCas13a), VI-B (PbuCas13b), VI-D (RfxCas13d), VI-X (Cas13x.1 Cas13x.2), VI-Y (Cas13y.1 Cas13y.2). A systematic evaluation effects on two transcripts (GhCLA GhPGF cotton) as well an virus (TMV tobacco) reveals that RfxCas13d, Cas13x.1, Cas13x.2 exhibit enhanced stability with efficiencies ranging 58 80%, closely followed by Cas13y.1 Cas13y.2. Notably, both Cas13x.1 can simultaneously degrade through tRNA-crRNA cassette approach, achieving up 50%. Furthermore, different enable varying degrees transcript minimal off-target effects, generating germplasms diverse mutant phenotypes. Transgenic tobacco plants show significant reductions damage, along mild oxidative stress accumulation viral particles after TMV infection. In conclusion, our study presents efficient reliable platform transcriptome holds promise plant functional research future crop improvement.

Язык: Английский

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mRNA m6A detection

Nature Reviews Methods Primers, Год журнала: 2024, Номер 4(1)

Опубликована: Дек. 12, 2024

Язык: Английский

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