Sensitive analysis of single nucleotide variation by Cas13d orthologs, EsCas13d and RspCas13d

Qiao, Xin; Gao, Yanmin; Li, Jiaojiao; Wang, Zhaoguan; Qiao, Hongyan; Qi, Hao

doi:10.1002/bit.27813

Cited by 12 publications

(5 citation statements)

References 34 publications

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“…The newly discovered Cas13d effector has shown several advantages over other Cas13 variants in terms of structure as well as in function: i) Its small size which makes Cas13d an ideal and compatible for packaging into a viral vector and carried-out efficient delivery in the mammalian system; ii) Neurotoxicity effect of Cas13d founds to be less or absent in mammalian system; iii) Cas13d has dual nuclease activity i.e., it can cleave targeted ssRNA and can process maturation of crRNA simultaneously without any requirement of additional helper ribonucleases and tracrRNA; iv) Cas13d does not require protospacer flanking sequence (PFS) for RNA cleavage activity and this makes it lesser sequence constraints and can be used efficiently in targeting of “strongly depleted arrays”; v) Cas13d does not show off-target transcriptional perturbations as compared to other Cas13 variants and exhibits higher efficiency and specificity in cleaving targeted RNA in the mammalian system compared to other Cas13 endonucleases of the Cas13 enzyme family. These superlative features of Cas13d make it a prominent candidate for spatiotemporal transcriptome engineering, nucleic acid detection, multiplex gene regulation, post-transcriptional gene silencing, alternative splicing, tracking, and RNA epigenetic regulation ( Yan et al, 2018 ; Wang et al, 2019 ; Zhang H. et al, 2019 ; Kumar et al, 2020 ; Liu et al, 2020 ; Qiao et al, 2021 ; Wu and Kapfhammer, 2021 ).…”

Section: Advantages Of Cas13d Over Other Cas13 Effectorsmentioning

confidence: 99%

“…(Rsp) and applied them to detect nucleic acid and they found that both the Cas13d exhibit efficient and rapid target RNA detection in picomolar range and even in the lower viral titer. On the whole, their study revealed that both EsCas13d and RspCas13d were able to detect the targeted RNA and even it can detect single-nucleotide variation with high sensitivity and specificity ( Qiao et al, 2021 ). Therefore, the CRISPR-Cas13d system can be utilized as a newly qualified molecular diagnostic tool to detect various pathogenic viruses and can also be used to identify biomarker genes for various diseases.…”

Section: Various Applications Of Cas13d In Basic Research and Biomedi...mentioning

confidence: 99%

See 1 more Smart Citation

Cas13d: A New Molecular Scissor for Transcriptome Engineering

Gupta¹,

Ghosh²,

Chakravarti³

et al. 2022

Front. Cell Dev. Biol.

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The discovery of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated Cas endonucleases in bacterial and archaeal species allowed scientists to modify, utilized, and revolutionize this tool for genetic alterations in any species. Especially the type II CRISPR-Cas9 system has been extensively studied and utilized for precise and efficient DNA manipulation in plant and mammalian systems over the past few decades. Further, the discovery of the type V CRISPR-Cas12 (Cpf1) system provides more flexibility and precision in DNA manipulation in prokaryotes, plants, and animals. However, much effort has been made to employ and utilize the above CRISPR tools for RNA manipulation but the ability of Cas9 and Cas12 to cut DNA involves the nuisance of off-target effects on genes and thus may not be employed in all RNA-targeting applications. Therefore, the search for new and diverse Cas effectors which can precisely detect and manipulate the targeted RNA begins and this led to the discovery of a novel RNA targeting class 2, type VI CRISPR-Cas13 system. The CRISPR-Cas13 system consists of single RNA-guided Cas13 effector nucleases that solely target single-stranded RNA (ssRNA) in a programmable way without altering the DNA. The Cas13 effectors family comprises four subtypes (a-d) and each subtype has distinctive primary sequence divergence except the two consensuses Higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN) that includes RNase motifs i.e. R-X4-6-H. These two HEPN domains are solely responsible for executing targetable RNA cleavage activity with high efficiency. Further, recent studies have shown that Cas13d exhibits higher efficiency and specificity in cleaving targeted RNA in the mammalian system compared to other Cas13 endonucleases of the Cas13 enzyme family. In addition to that, Cas13d has shown additional advantages over other Cas13 variants, structurally as well as functionally which makes it a prominent and superlative tool for RNA engineering and editing. Therefore considering the advantages of Cas13d over previously characterized Cas13 subtypes, in this review, we encompass the structural and mechanistic properties of type VI CRISPR-Cas13d systems, an overview of the current reported various applications of Cas13d, and the prospects to improve Cas13d based tools for diagnostic and therapeutic purposes.

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Section: Advantages Of Cas13d Over Other Cas13 Effectorsmentioning

confidence: 99%

Section: Various Applications Of Cas13d In Basic Research and Biomedi...mentioning

confidence: 99%

Cas13d: A New Molecular Scissor for Transcriptome Engineering

Gupta¹,

Ghosh²,

Chakravarti³

et al. 2022

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

show abstract

“…Type V and VI CRISPR-Cas systems such as Cas12a, Cas12b, and Cas13a-d have been harnessed for the detection of nucleic acids 1,[22][23][24][25] . While Cas12a and Cas13a have been extensively deployed for on-site applications, Cas12b has recently emerged as a distinct class of enzyme, the majority of which are thermostable [26][27][28] .…”

Section: Identification Of Stabilizing Mutants Of Brcas12bmentioning

confidence: 99%

Engineering Highly Thermostable Cas12b via De Novo Structural Analyses for One-Pot Detection of Nucleic Acids

Nguyễn

Rananaware

Yang

et al. 2022

Preprint

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostics have elevated nucleic acid detection in terms of sensitivity, specificity, and rapidity in recent years. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify workflow and reduce carryover contamination. Here, we report an engineered Cas12b system from Brevibacillus (eBrCas12b) with improved thermostability that falls within the optimal range (60-65°C) of the Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP). Using de novo structural analyses via DeepDDG and HotSpot Wizard based on Alpha Fold and SWISS-MODEL predicted structures, mutations were introduced into the REC and RuvC domains of wild-type BrCas12b to tighten the hydrophobic cores of the protein, thereby enhancing its stability at high temperatures. We expressed, purified, and systematically characterized 49 BrCas12b variants with the emphasis on functionality and thermostability. The assay utilizing eBrCas12b, which we coined SPLENDID (Single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting-4°C and 7°C higher than wild-type BrCas12b and AapCas12b, respectively. We further validated SPLENDID clinically in 40 Hepatitis C (HCV) positive and 40 negative serum samples. A specificity of 97.5%, an accuracy of 90.0%, and a sensitivity of 82.5% were achieved. Results can be obtained via one-pot testing in as little as 20 minutes. With the extraction process, the entire assay can be performed in under an hour. Therefore, we believe that SPLENDID has the potential to become a widely universal platform for the detection of infectious diseases.

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“…As a result, Cas13d has exhibited great potential in gene knockdown, [10] antiviral defense both in cells and animals, [11] and more recently, viral RNA detection [12] . Although promising, Cas13d‐based diagnostics is still in its infancy where its diagnostic utility is limited to detect nucleic‐acid‐related biomarkers [13] . To explore new dimensions in Cas13d‐based diagnostics, there is still a need to combine Cas13d‐based technologies with other signal transduction strategies to construct an intelligent sensing system for sensitive detection of non‐nucleic‐acid biomarkers such as proteins.…”

Section: Introductionmentioning

confidence: 99%

Harnessing the CRISPR‐Cas13d System for Protein Detection by Dual‐Aptamer‐Based Transcription Amplification

Feng

Liu

et al. 2023

Chemistry A European J

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CRISPR-based biosensing technology has been emerging as a revolutionary diagnostic tool for many diseaserelated biomarkers. In particular, RspCas13d, a newly identified RNA-guided Cas13d ribonuclease derived from Ruminococcus sp., has shown great promise for accurate and sensitive detection of RNA due to its RNA sequence-specific recognition and robust collateral trans-cleavage activity. However, its diagnostic utility is limited to detecting nucleic-acidrelated biomarkers. To address this limitation, herein we present a proof-of-concept demonstration of a target-responsive CRISPR-Cas13d sensing system for protein biomarkers. This system was rationally designed by integrating a dual-aptamer-based transcription amplification strategy with CRISPR-Cas13d (DATAS-Cas13d), in which the protein binding initiates in-vitro RNA transcription followed by the activation of RspCas13d. Using a short fluorescent ssRNA as the signal reporter and cardiac troponin I (cTnI) as the model analyte, the DATAS-Cas13d system showed a wide linear range, low detection limit, and high specificity for the detection of cTnI in buffer and human serum. Thanks to the facile integration of various bioreceptors into the DATAS-Cas13d system, the method could be adapted to detecting a broad range of clinically relevant protein biomarkers, and thus broaden the medical applications of Cas13d-based diagnostics.

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Sensitive analysis of single nucleotide variation by Cas13d orthologs, EsCas13d and RspCas13d

Cited by 12 publications

References 34 publications

Cas13d: A New Molecular Scissor for Transcriptome Engineering

Cas13d: A New Molecular Scissor for Transcriptome Engineering

Engineering Highly Thermostable Cas12b via De Novo Structural Analyses for One-Pot Detection of Nucleic Acids

Harnessing the CRISPR‐Cas13d System for Protein Detection by Dual‐Aptamer‐Based Transcription Amplification

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