Definition of CRISPR Cas12a Trans-Cleavage Units to Facilitate CRISPR Diagnostics

Lv, Hailong; Wang, Jian; Zhang, Jian; Chen, Yi-Jian; Yin, Lei; Jin, Dian; Gu, Dayong; Zhao, Huichan; Xu, Yong; Wang, Jin

doi:10.3389/fmicb.2021.766464

Cited by 48 publications

(55 citation statements)

References 22 publications

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“…In addition to changing the divalent cations in the reaction buffer, the addition of additives can also improve the sensitivity of Cas12 system. Polyethylene glycol (PEG), glycerin and Triton X-100 can improve the sensitivity of Cas12a system, but their mechanism of promoting trans-cleavage activity is unknown [ 16 ]. Li et al [ 17 ] found that bovine serum albumin (BSA) could improve sensitivity of Cas12a system, while L-proline could protect Cas12a from an adverse environment.…”

Section: Strategies For Improving Sensitivitymentioning

confidence: 99%

“…Yue et al [ 15 ] found that LbCas12a had the highest trans-cleavage activity under weakly alkaline conditions (pH 9). Lv et al [ 16 ] analyzed the trans-cleavage activity of LbCas12a in reaction buffers with pH ranging from 3.5 to 9.6 in increments of 0.1. Among the tested buffers, LbCas12a preferred buffers with pH 8.5 and 8.6.…”

Section: Strategies For Improving Sensitivitymentioning

confidence: 99%

“…Among the tested buffers, LbCas12a preferred buffers with pH 8.5 and 8.6. Currently, commercial NEBuffer 2.1 and NEBuffer 3.1 are often selected as reaction buffers for Cas12 in many studies, but optimized buffers in some studies can achieve a more sensitive Cas12 system than these two commercial buffers [ 14 – 16 , 18 , 19 ]. In general, the divalent cation, additive and pH are three important conditions that affect the sensitivity of the Cas12 system (Table 1 summarizes the components of the reaction buffers mentioned in this paragraph).…”

Section: Strategies For Improving Sensitivitymentioning

confidence: 99%

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Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection

2022

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The COVID-19 pandemic has brought great challenges to traditional nucleic acid detection technology. Thus, it is urgent to develop a more simple and efficient nucleic acid detection technology. CRISPR-Cas12 has signal amplification ability, high sensitivity and high nucleic acid recognition specificity, so it is considered as a nucleic acid detection tool with broad development prospects and high application value. This review paper discusses recent advances in CRISPR-Cas12-based nucleic acid detection, with an emphasis on the new research methods and means to improve the nucleic acid detection capability of CRISPR-Cas12. Strategies for improving sensitivity, optimization of integrated detection, development of simplified detection mode and improvement of quantitative detection capabilities are included. Finally, the future development of CRISPR-Cas12-based nucleic acids detection is prospected.

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Section: Strategies For Improving Sensitivitymentioning

confidence: 99%

Section: Strategies For Improving Sensitivitymentioning

confidence: 99%

Section: Strategies For Improving Sensitivitymentioning

confidence: 99%

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Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection

2022

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“…Yu and colleagues created an all-in-one TA test using the telomere-synthesis-induced CRISPR-Cas12a system [ 71 ]. Extension of the telomere by telomerase resulted in telomere repeats (TTAGGG)n, which could be recognized using a customized CRISPR-guided RNA (crRNA) and activated by the conventional trans-cleavage CRISPR- Cas12a detection assay [ 72 ]. Because CRISPR-Cas12a is so sensitive, this method achieved a linear regression spanning 100 to 2000 HeLa cells and a detection limit of only 26 HeLa cells [ 71 ].…”

Section: Targeting Telomerase By Crispr/casmentioning

confidence: 99%

CRISPR/Cas: A New Tool in the Research of Telomeres and Telomerase as Well as a Novel Form of Cancer Therapy

Porika

Tippani

Saretzki

2022

IJMS

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Due to their close connection with senescence, aging, and disease, telomeres and telomerase provide a unique and vital research route for boosting longevity and health span. Despite significant advances during the last three decades, earlier studies into these two biological players were impeded by the difficulty of achieving real-time changes inside living cells. As a result of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated system’s (Cas) method, targeted genetic studies are now underway to change telomerase, the genes that govern it as well as telomeres. This review will discuss studies that have utilized CRISPR-related technologies to target and modify genes relevant to telomeres and telomerase as well as to develop targeted anti-cancer therapies. These studies greatly improve our knowledge and understanding of cellular and molecular mechanisms that underlie cancer development and aging.

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“…The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) systems have been considered to have great potential in diagnostic, thus revolutionizing the nucleic acid detection area (Wang et al, 2020b;Lv et al, 2021). The most commonly used Cas proteins in the CRISPR/Cas-based nucleic acid detection system are Cas9, Cas12, Cas13 and Cas14 (Wang et al, 2020e).…”

Section: Introductionmentioning

confidence: 99%

Rapid and Specific Detection of Active SARS-CoV-2 With CRISPR/Cas12a

Liu

Wang

et al. 2022

Front. Microbiol.

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Rapid and sensitive nucleic acid detection of SARS-CoV-2 has contributed to the clinical diagnosis and control of COVID-19. Although detection of virus genomic RNA (gRNA) has been commonly used in clinical diagnosis, SARS-CoV-2 gRNA detection could not discriminate between active infectious virus with remnant viral RNA. In contrast to genomic RNA, subgenomic RNAs (sgRNAs) are only produced when the virus is actively replicating and transcription, detection of sgRNA could be an indication to evaluate infectivity. CRISPR/Cas-based nucleic acid detection methods have been considered potential diagnostic tools due to their intrinsic sensitivity, specificity and simplicity. In this study, to specifically detect active virus replication, we developed a CRISPR-based active SARS-CoV-2 (CRISPR-actCoV) detection strategy by detecting sgRNAs of SARS-CoV-2. CRISPR-actCoV with CRISPR Cas12a-assisted fluorescence reporter system enables detection of sgRNAs at 10 copies in 35 min with high specificity and can be read out with naked eyes. Further, we performed CRISPR-actCoV mediated sgRNA detection in 30 SARS-CoV-2 potentially infected clinical samples, and 21 samples were SARS-CoV-2 sgRNA positive. A quantitative RT-PCR assay was also performed to detect gRNA of SARS-CoV-2 in parallel. Among the 30 clinical samples, 27 samples were gRNA positive. Taken together, CRISPR-actCoV provides an alternative for rapid and accurate detection of active SARS-CoV-2 and has great significance in better response of coronavirus causing epidemic disease.

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Definition of CRISPR Cas12a Trans-Cleavage Units to Facilitate CRISPR Diagnostics

Cited by 48 publications

References 22 publications

Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection

Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection

CRISPR/Cas: A New Tool in the Research of Telomeres and Telomerase as Well as a Novel Form of Cancer Therapy

Rapid and Specific Detection of Active SARS-CoV-2 With CRISPR/Cas12a

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