2′-<i>O</i>-Methyl modified guide RNA promotes the single nucleotide polymorphism (SNP) discrimination ability of CRISPR–Cas12a systems

Ke, Yuqing; Ghalandari, Behafarid; Huang, Shiyi; Li, Sijie; Huang, Chengjie; Zhi, Xiao; Cui, Daxiang; Li, Yiyang

doi:10.1039/d1sc06832f

Cited by 33 publications

(21 citation statements)

References 66 publications

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“…CRISPR/Cas12a, an RNA-guided DNase, demonstrates ssDNase ability (also called collateral effect or collateral cleavage) when the Cas12a/crRNA complex is activated by the target dsDNA. − In the CRISPR/Cas12a detection system, ssDNA is usually dual-labeled with a fluorophore and quencher so that the dsDNA input can be transformed into the measurable fluorescent output. ,, CRISPR/Cas12a is advantageous for being both temperature-resilient and highly specific . Cas12a can be activated even at room temperature and performs low mismatch tolerance. , These features enable the CRISPR/Cas12a system to be an efficient and programable biosensing platform. − However, the CRISPR/Cas12a system alone is challenged for its limited sensitivity. , A typical CRISPR/Cas12a system can hardly provide detectable signals when the concentration of the dsDNA substrate is lower than 100 pM .…”

Section: Introductionmentioning

confidence: 99%

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Harnessing Multiplex crRNA in the CRISPR/Cas12a System Enables an Amplification-Free DNA Diagnostic Platform for ASFV Detection

Zeng

Zhuang

et al. 2022

Anal. Chem.

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CRISPR-associated (Cas) protein systems have been increasingly incorporated in nucleic-acid diagnosis. CRISPR/ Cas12a can cleave single-stranded DNA (ssDNA) after being guided to the target double-stranded DNA (dsDNA) with crRNA, making it a specific tool for dsDNA detection. Assisted by nucleic acid preamplification, CRISPR/Cas12a enables dsDNA detection at the attomolar level. However, such mandatory preamplification in CRISPR/Cas12a also accompanies the extra step of transferring preamplification products into the CRISPR/Cas12a system, which is not only cumbersome and time-consuming but also induces the risk of cross-contamination. Herein, we demonstrate a multiplex-crRNA strategy to enhance the sensitivity of the CRISPR/Cas12a system without any preamplification. This multiplex-crRNA strategy harnesses multiple sequences of crRNA which target different regions of the same dsDNA substrate in the same CRISPR/Cas12a system. Therefore, detection signals are accumulated without amplification, which augments the conventional detection limit. For application demonstration, the B646L gene from the African swine fever virus (ASFV), which is a dsDNA virus, is exemplified. The detection limit of the multiplex-crRNA system can be improved to ∼1 picomolar (pM) without amplification, which is ∼64 times stronger than the conventional single-crRNA system. The multiplex-crRNA system presented in this study, with slight modifications, can be generalized to other biosensing settings where preamplification is not readily available.

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Section: Introductionmentioning

confidence: 99%

“…1,5,6 CRISPR/Cas12a is advantageous for being both temperature-resilient and highly specific. 7 Cas12a can be activated even at room temperature and performs low mismatch tolerance. 5,8 These features enable the CRISPR/Cas12a system to be an efficient and programable biosensing platform.…”

Section: ■ Introductionmentioning

confidence: 99%

Harnessing Multiplex crRNA in the CRISPR/Cas12a System Enables an Amplification-Free DNA Diagnostic Platform for ASFV Detection

Zeng

Zhuang

et al. 2022

Anal. Chem.

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“…Hence, nanotechnology-based drug delivery platforms, especially drug-containing NPs offer better pharmacokinetic parameters. For example, the platforms improve the accumulation of drugs in the tumor site through enhanced permeability and retention (EPR) [ 16 ].…”

Section: Biological Barriersmentioning

confidence: 99%

“…After the creation of the Precision Medicine Initiative (PMI) in 2015 [ 14 ], precision medicine put emphasis on tailoring specific treatment plan to individual by accounting for multiple genetic and epigenetic characteristics [ 15 ]. Nanotechnologies for drug delivery are designed to overcome heterogeneity among patients because of their engineering functional and structural properties, which significantly improves drug specificity, optimizes dosing, and combinatorial strategies [ 16 , 17 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

Precise Design Strategies of Nanotechnologies for Controlled Drug Delivery

Huang

2022

JFB

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Rapid advances in nanotechnologies are driving the revolution in controlled drug delivery. However, heterogeneous barriers, such as blood circulation and cellular barriers, prevent the drug from reaching the cellular target in complex physiologic environments. In this review, we discuss the precise design of nanotechnologies to enhance the efficacy, quality, and durability of drug delivery. For drug delivery in vivo, drugs loaded in nanoplatforms target particular sites in a spatial- and temporal-dependent manner. Advances in stimuli-responsive nanoparticles and carbon-based drug delivery platforms are summarized. For transdermal drug delivery systems, specific strategies including microneedles and hydrogel lead to a sustained release efficacy. Moreover, we highlight the current limitations of clinical translation and an incentive for the future development of nanotechnology-based drug delivery.

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“…Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease (CRISPR/Cas) systems have been developed to detect nucleic acid, including Cas13a (20)(21)(22)(23)(24)(25)(26), Cas12a (27)(28)(29)(30)(31), Cas9 (32)(33)(34), Cas12b (35), and Cas14 (36). Cas12a, an RNA-guided DNA endonuclease, recognizes a T nucleotide rich, such as 5…”

Section: Introductionmentioning

confidence: 99%

One-Pot Visual Detection of African Swine Fever Virus Using CRISPR-Cas12a

Qin

Zhu

et al. 2022

Front. Vet. Sci.

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African swine fever virus (ASFV) is a leading cause of worldwide agricultural loss. ASFV is a highly contagious and lethal disease for both domestic and wild pigs, which has brought enormous economic losses to a number of countries. Conventional methods, such as general polymerase chain reaction and isothermal amplification, are time-consuming, instrument-dependent, and unsatisfactorily accurate. Therefore, rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control. Herein, we created a one-pot visual detection system for ASFV with CRISPR/Cas12a technology combined with LAMP or RPA. A mineral oil sealing strategy was adopted to mitigate sample cross-contamination between parallel vials during high-throughput testing. Furthermore, the blue fluorescence signal produced by ssDNA reporter could be observed by the naked eye without any dedicated instrument. For CRISPR-RPA system, detection could be completed within 40 min with advantageous sensitivity. While CRISPR-LAMP system could complete it within 60 min with a high sensitivity of 5.8 × 102 copies/μl. Furthermore, we verified such detection platforms display no cross-reactivity with other porcine DNA or RNA viruses. Both CRISPR-RPA and CRISPR-LAMP systems permit highly rapid, sensitive, specific, and low-cost Cas12a-mediated visual diagnostic of ASFV for point-of-care testing (POCT) applications.

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2′-O-Methyl modified guide RNA promotes the single nucleotide polymorphism (SNP) discrimination ability of CRISPR–Cas12a systems

Abstract: This study illustrates that 2′-O-methyl modified gRNAs improve the specificity of the CRISPR–Cas12a system (mg-CRISPR) via suppressing the Cas12a's affinity to off-target DNA and provides an efficient strategy for high-specificity gRNA design.

Cited by 33 publications

References 66 publications

Harnessing Multiplex crRNA in the CRISPR/Cas12a System Enables an Amplification-Free DNA Diagnostic Platform for ASFV Detection

Harnessing Multiplex crRNA in the CRISPR/Cas12a System Enables an Amplification-Free DNA Diagnostic Platform for ASFV Detection

Precise Design Strategies of Nanotechnologies for Controlled Drug Delivery

One-Pot Visual Detection of African Swine Fever Virus Using CRISPR-Cas12a

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