Article
Biophysics
Liangfen Cheng, Fuhan Yang, Yining Zhao, Ziye Liu, Xudong Yao, Juan Zhang
Summary: This study utilizes a tetrahedron supported CRISPR/Cas13a cleavage method for direct electrochemical detection of circular RNA (circRNA) in urine from bladder cancer patients. The CRISPR/Cas13a system is designed to recognize the characteristic back-splice junction site of circRNA. The activated CRISPR/Cas13a cleaves uracil bases in a DNA tetrahedron immobilized on a gold electrode, resulting in the release of an electrochemical active molecule, methylene blue. The developed electrochemical biosensor demonstrates high catalytic efficiency and a rigid structure, allowing for direct detection of circRNA in 25 μL urine samples with a lowest detection limit of 0.089 fM and a linear detection range from 10 fM to 50 nM in less than 10 minutes, avoiding the need for complicated extraction and enabling on-site detection.
BIOSENSORS & BIOELECTRONICS
(2023)
Article
Biophysics
Xiao-Min Hang, Peng-Fei Liu, Sen Tian, Hui-Yi Wang, Kai -Ren Zhao, Li Wang
Summary: This study reports a fast and simple Cas13a-based assay for direct detection of Ebola RNA in unamplified samples. The method combines a 10-min Cas13a-mediated cleavage step with a 30-min DNA roller amplification step, achieving a low limit of detection and excellent anti-interfering performance. Additionally, the method can be completed in 40 minutes without any pre-processing steps.
BIOSENSORS & BIOELECTRONICS
(2022)
Article
Medicine, Research & Experimental
Lin Wang, Junhu Zhou, Qixue Wang, Yunfei Wang, Chunsheng Kang
Summary: This study proposes a potential anti-SARS-CoV-2 strategy based on the CRISPR-Cas13a system, identifying a specific RNA segment of the spike protein and designing an efficient crRNA sequence for cleavage. It provides a rapid design pipeline for an antiviral tool against SARS-CoV-2 and introduces a novel approach for anti-virus study.
Article
Biophysics
Ting Zhang, Wenhu Zhou, Xiaoya Lin, Mohammad Rizwan Khan, Sha Deng, Mi Zhou, Guiping He, Chengyong Wu, Ruijie Deng, Qiang He
Summary: This study developed a new detection system that can accurately distinguish viable pathogenic bacteria without the need for reverse transcription, nucleic acid amplification, or labeled substrates. By utilizing CRISPR-Cas13a and a light-up RNA aptamer, it enables rapid detection and precise quantification of viable pathogens.
BIOSENSORS & BIOELECTRONICS
(2021)
Article
Food Science & Technology
Song Gao, Jingwen Liu, Zhiyong Li, Yi Ma, Jufang Wang
Summary: This study proposed a PCF detection method based on the CRISPR-Cas13a system for sensitive and specific detection of Salmonella, showing excellent sensitivity to detect Salmonella genomic DNA with a minimum of 10(1) aM or 10 degrees CFU/ml Salmonella bacteria within 2 hours. The method also demonstrated good specificity with no cross-reaction with other common foodborne bacteria.
JOURNAL OF FOOD SCIENCE
(2021)
Article
Engineering, Biomedical
Lin Liu, Zheyu Wang, Yixuan Wang, Jingyi Luan, Jeremiah J. Morrissey, Rajesh R. Naik, Srikanth Singamaneni
Summary: This study demonstrates a novel, ultrasensitive, and amplification-free RNA quantification method by integrating CRISPR-Cas13a system with a plasmonic nanolabel. The method shows great potential in quantitative monitoring of cancer biomarkers and provides a rapid, sensitive strategy for detection of various RNA biomarkers, even in resource-limited settings.
ADVANCED HEALTHCARE MATERIALS
(2021)
Article
Chemistry, Analytical
Pengfei Song, Pengbo Zhang, Ke Qin, Fengxia Su, Kejian Gao, Xin Liu, Zhengping Li
Summary: This study utilizes CRISPR/Cas13a to recognize the unique junction sequence of circRNA and develops a CRISPR/Cas13a induced exponential amplification assay for sensitive and specific detection of circRNA. The designed primers and high amplification efficiency allow for the detection of low concentrations of circRNA in real biological samples.
Article
Chemistry, Multidisciplinary
Tian Tian, Bowen Shu, Yongzhong Jiang, Miaomiao Ye, Lei Liu, Zhonghui Guo, Zeping Han, Zhang Wang, Xiaoming Zhou
Summary: The confinement effect-inspired Cas13a assay for single-molecule RNA diagnostics eliminates the need for nucleic acid amplification and reverse transcription, achieving a >10,000-fold enhancement in sensitivity and enabling absolute digital single-molecule RNA quantitation. Its broad applicability and accurate detection of various RNA molecules at the single-molecule level make it a potential dominant rival to RT-qPCR.
Article
Chemistry, Analytical
Yuxi Wang, Ting Xue, Minjin Wang, Rodrigo Ledesma-Amaro, Ying Lu, Xinyue Hu, Ting Zhang, Ming Yang, Yalun Li, Jin Xiang, Ruijie Deng, Binwu Ying, Weimin Li
Summary: In this study, a CRISPR-Cas13a cascade-based viral RNA assay was developed for label-free and isothermal detection of SARS-CoV-2 and its mutations in clinical samples. The assay demonstrated high specificity to distinguish SARS-CoV-2 from other coronaviruses and high sensitivity with a limit of detection of 0.216 fM. It also showed the capability to discriminate single-nucleotide mutations. The validation results showed 100% agreement with RT-qPCR. This assay has the potential to be used as a routine nucleic acid test for SARS-CoV-2 in resource-limited regions.
SENSORS AND ACTUATORS B-CHEMICAL
(2022)
Article
Chemistry, Analytical
Dan Zhao, Jiutang Tang, Qin Tan, Xiaohong Xie, Xin Zhao, Dingpei Xing
Summary: In this study, a dual-amplified biosensing method based on CRISPR/Cas13a-triggered Cas12a, namely Cas13a-12a amplification, was developed for miRNA-155 detection. The detection limit of this amplification strategy was as low as 0.35 fM, which was much lower than that of the only Cas13a-based assay. Accurate quantification of miRNA-155 expression levels in different cancer patients was achieved using this strategy. Hence, the developed method provides a supersensitive and highly specific miRNA sensing platform for clinical applications.
Article
Biophysics
Wenjun Liu, Xuena Zhu, Ling Li, Shanshan Wang, Chen-Zhong Li, Tingbo Liang
Summary: Activation of Cas13 is dependent on crRNA-target RNA hybridization. This study develops a controlled activation system for Cas13 using N-terminus tagging, which suppresses target dependent activation through a composite SUMO tag. This discovery not only enables Cas13-based protease detection, but also provides a novel strategy for precise multi-component activation of Cas13.
BIOSENSORS & BIOELECTRONICS
(2023)
Article
Engineering, Environmental
Jihua Wei, Zichun Song, Jiuying Cui, Yuanxun Gong, Qianli Tang, Kai Zhang, Xinlei Song, Xianjiu Liao
Summary: In this study, an electrochemiluminescence (ECL) sensing platform based on the Entropy-driven triggered T7 amplification-CRISPR/Cas13a system (EDT-Cas) was introduced. The platform combines a programmable entropy-driven cycling strategy, T7 RNA polymerase, and the CRISPR/Cas13a system to amplify the determination of the SARS-CoV-2 RdRp gene. The Ti3C2Tx-compliant ECL signaling molecule offers unique benefits when used with the ECL sensing platform to increase the assay sensitivity and the electrode surface modifiability.
JOURNAL OF HAZARDOUS MATERIALS
(2023)
Article
Chemistry, Analytical
Yixia Yang, Wenfu Yi, Feng Gong, Zhiyou Tan, Xiaoyun Shan, Jingjing Qian, Conghua Xie, Xinghu Ji, Zhenhua Zheng, Zhike He
Summary: This study presented a new diagnostic assay, named AIOC, based on All-In-One CRISPR/Cas13a and a DNA circuit for detecting ECHO 11. The simplified assay combined CRISPR/Cas13a and catalytic hairpin assembly (CHA) circuit in one-pot reaction, enabling effective avoidance of multiple handling steps and uncapping contamination. The AIOC assay demonstrated rapid, isothermal, ultrasensitive, specific, user-friendly performance without the need for RNA reverse-transcription.
SENSORS AND ACTUATORS B-CHEMICAL
(2023)
Article
Biochemistry & Molecular Biology
Martin Pacesa, Chun-Han Lin, Antoine Clery, Aakash Saha, Pablo R. Arantes, Katja Bargsten, Matthew J. Irby, Frederic H. -T. Allain, Giulia Palermo, Peter Cameron, Paul D. Donohoue, Martin Jinek
Summary: The crystallographic structures of Cas9 bound to off-target substrates reveal that noncanonical base-pairing interactions within the guide:off-target heteroduplex enable off-target binding. Single-nucleotide deletions in off-target substrates are accommodated by base skipping or multiple noncanonical base pairs. PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe. These insights contribute to the improved rational design of guide RNAs and off-target prediction algorithms.
Article
Biochemistry & Molecular Biology
Leila Kashefi-Kheyrabadi, Huynh Vu Nguyen, Anna Go, Min-Ho Lee
Summary: This study presents a CRISPR/Cas13a-powered electrochemical multiplexed biosensor for detecting SARS-CoV-2 RNA strands. It offers a multiplexed and NAA-free strategy for detecting SARS-CoV-2 RNA fragments, achieving low detection limits and demonstrating specificity, simplicity, and universality. The biosensor has the potential to rival RT-PCR as a diagnostic method for SARS-CoV-2.
BIOELECTROCHEMISTRY
(2023)
Editorial Material
Biochemistry & Molecular Biology
Brandon J. Davis, Mitchell R. O'Connell
Article
Biochemistry & Molecular Biology
Simon Eitzinger, Amina Asif, Kyle E. Watters, Anthony T. Iavarone, Gavin J. Knott, Jennifer A. Doudna, Fayyaz ul Amir Afsar Minhas
NUCLEIC ACIDS RESEARCH
(2020)
Article
Chemistry, Multidisciplinary
Jing Dai, Gavin J. Knott, Wen Fu, Tiffany W. Lin, Ariel L. Furst, R. David Britt, Matthew B. Francis
Summary: The artificial multiheme system based on cpTMV shows structural and functional similarity to natural multiheme cytochrome c proteins. The mutant Q101H creates a circular assembly of 17 protein-embedded hemes, providing further engineering opportunities for tuning the redox properties of cofactors and incorporating non-native components. Emulating electron transfer pathways in nature using a tunable artificial system can contribute to the development of photocatalytic materials and bioelectronics.
Article
Biochemistry & Molecular Biology
Parinaz Fozouni, Sungmin Son, Maria Diaz de Leon Derby, Gavin J. Knott, Carley N. Gray, Michael D'Ambrosio, Chunyu Zhao, Neil A. Switz, G. Renuka Kumar, Stephanie Stephens, Daniela Boehm, Chia-Lin Tsou, Jeffrey Shu, Abdul Bhuiya, Maxim Armstrong, Andrew R. Harris, Pei-Yi Chen, Jeannette M. Osterloh, Anke Meyer-Franke, Bastian Joehnk, Keith Walcott, Anita Sil, Charles Langelier, Katherine S. Pollard, Emily D. Crawford, Andreas S. Puschnik, Maira Phelps, Amy Kistler, Joseph L. DeRisi, Jennifer A. Doudna, Daniel A. Fletcher, Melanie Ott
Summary: This study developed an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2, showing rapid, highly sensitive, and mobile phone-based readout capabilities.
Review
Biochemistry & Molecular Biology
Haridha Shivram, Brady F. Cress, Gavin J. Knott, Jennifer A. Doudna
Summary: CRISPR-Cas systems are widely used for genome editing and therapeutics, but controlling the timing, duration, efficiency, and specificity of enzyme activities is crucial for their full potential. This review discusses natural biomolecules that regulate CRISPR-Cas immunity and the engineering strategies to enhance or inhibit enzyme function, as well as their applications in CRISPR biology and technologies. Unanswered questions about the evolution and purpose of these CRISPR regulators are highlighted in this review.
NATURE CHEMICAL BIOLOGY
(2021)
Article
Multidisciplinary Sciences
Alexandra M. Amen, Christof Fellmann, Katarzyna M. Soczek, Shawn M. Ren, Rachel J. Lew, Gavin J. Knott, Jesslyn E. Park, Andrew M. McKinney, Andrew Mancini, Jennifer A. Doudna, Joseph F. Costello
Summary: Mutations in the TERT promoter of glioblastomas create binding sites for GABP transcription factor complexes, leading to reactivation of TERT and maintenance of telomeres. GBM cells with TERT promoter mutations display a dependence on GABPB1L for proliferation, which can be rescued by upregulation of the protein paralogue GABPB2. Inhibition of GABPB1L in combination with chemotherapy results in reduced growth of intracranial GBM tumors by impairing DNA damage response.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
(2021)
Article
Biochemistry & Molecular Biology
Tina Y. Liu, Gavin J. Knott, Dylan C. J. Smock, John J. Desmarais, Sungmin Son, Abdul Bhuiya, Shrutee Jakhanwal, Noam Prywes, Shreeya Agrawal, Maria Diaz de Leon Derby, Neil A. Switz, Maxim Armstrong, Andrew R. Harris, Emeric J. Charles, Brittney W. Thornton, Parinaz Fozouni, Jeffrey Shu, Stephanie Stephens, G. Renuka Kumar, Chunyu Zhao, Amanda Mok, Anthony T. Iavarone, Arturo M. Escajeda, Roger McIntosh, Shin E. Kim, Eli J. Dugan, Katherine S. Pollard, Ming X. Tan, Melanie Ott, Daniel A. Fletcher, Liana F. Lareau, Patrick D. Hsu, David F. Savage, Jennifer A. Doudna
Summary: The direct, amplification-free detection of RNA through CRISPR nucleases enables rapid and sensitive RNA detection, particularly suitable for point-of-care infection diagnosis.
NATURE CHEMICAL BIOLOGY
(2021)
Article
Multidisciplinary Sciences
Eric A. Nalefski, Nidhi Patel, Philip J. Y. Leung, Zeba Islam, Remy M. Kooistra, Ishira Parikh, Estelle Marion, Gavin J. Knott, Jennifer A. Doudna, Anne-Laure M. Le Ny, Damian Madan
Summary: The Cas13a and Cas12a enzymes in bacterial CRISPR systems exhibit distinct activity characteristics, with Cas13a showing high-turnover trans-RNase activity and Cas12a showing low-turnover trans-nuclease activity.
Article
Biochemistry & Molecular Biology
Gavin J. Knott, Yee Seng Chong, Daniel M. Passon, Xue-Hai Liang, Evelyne Deplazes, Maria R. Conte, Andrew C. Marshall, Mihwa Lee, Archa H. Fox, Charles S. Bond
Summary: DBHS proteins, including NONO and PSPC1, form different dimers and play important roles in RNA/DNA binding. The study reveals selectivity between different dimers and cooperative RNA recognition mechanisms, shedding light on how these proteins recognize a broad spectrum of RNA targets.
NUCLEIC ACIDS RESEARCH
(2022)
Article
Biochemistry & Molecular Biology
Ethan J. Walker, John Q. Bettinger, Kevin A. Welle, Jennifer R. Hryhorenko, Adrian M. Molina Vargas, Mitchell R. O'Connell, Sina Ghaemmaghami
Summary: This study provides proteome-wide evidence that the thermodynamic folding stability of proteins strongly influences the oxidation rates of buried methionine residues. These findings highlight the utility of using oxidation rates as a metric of folding stability and suggest a correlation between folding stability and optimal growth temperatures.
JOURNAL OF BIOLOGICAL CHEMISTRY
(2022)
Article
Biochemistry & Molecular Biology
Joshua C. Cofsky, Katarzyna M. Soczek, Gavin J. Knott, Eva Nogales, Jennifer A. Doudna
Summary: Researchers have discovered that the Cas9 protein bends and twists DNA when binding to PAM, allowing DNA nucleotides to be extracted from the double helix and examined by the guide RNA. This finding is significant for understanding the efficiency of genome editing.
NATURE STRUCTURAL & MOLECULAR BIOLOGY
(2022)
Article
Biochemistry & Molecular Biology
Pei Wen Lee, Andrew C. Marshall, Gavin J. Knott, Simon Kobelke, Luciano Martelotto, Ellie Cho, Paul J. McMillan, Mihwa Lee, Charles S. Bond, Archa H. Fox
Summary: The RNA-binding proteins of the DBHS family, NONO, SFPQ, and PSPC1, play important roles in genome stability and transcriptional regulation. This study reveals that the dimerization of DBHS proteins influences their localization and function. The results show that NONO and SFPQ can form dimers and partner swapping between them is crucial for the subnuclear distribution of NONO.
JOURNAL OF BIOLOGICAL CHEMISTRY
(2022)
Article
Multidisciplinary Sciences
Arica R. VanderWal, Jung-Un Park, Bogdan Polevoda, Julia K. Nicosia, Adrian M. Molina Vargas, Elizabeth H. Kellogg, Mitchell R. O'Connell
Summary: In this study, researchers discovered that the membrane protein Csx28 in Type VI CRISPR-Cas systems enhances antiviral defense by slowing cellular metabolism upon viral infection. The antiviral activity of Csx28 requires sequence-specific cleavage of viral messenger RNAs by Cas13b. High-resolution cryo-electron microscopy revealed that Csx28 forms an octameric pore-like structure, which localizes to the inner membrane in vivo.
Article
Biochemistry & Molecular Biology
Souvik Sinha, Adrian M. Molina Vargas, Pablo R. Arantes, Amun Patel, Mitchell R. O'Connell, Giulia Palermo
Summary: Cas13a is a recent addition to the CRISPR-Cas toolkit that specifically targets RNA for detection purposes. This study reveals that target RNA acts as an allosteric effector, amplifying communication signals to control RNA cleavage activity. The findings provide a fundamental understanding of the mechanism of action of Cas13a and open up new avenues for the development of highly selective RNA-based cleavage and detection tools.
NUCLEIC ACIDS RESEARCH
(2023)
Article
Biochemistry & Molecular Biology
Adrian M. Molina Vargas, Souvik Sinha, Raven Osborn, Pablo R. Arantes, Amun Patel, Stephen Dewhurst, Dwight J. Hardy, Andrew Cameron, Giulia Palermo, Mitchell R. O'Connell
Summary: The increasing demand for sensitive, fast, and inexpensive nucleic acid-based detection technologies in clinical diagnostics has led to the successful application of RNA-guided ribonuclease CRISPR-Cas13. However, detecting genetic variations, such as single-nucleotide polymorphisms, remains challenging. In this study, the researchers expanded their understanding of LbuCas13a RNA detection specificity and developed novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. These advancements allowed for superior discrimination of SARS-CoV-2 strains compared to the wild-type LbuCas13a. These findings provide new design criteria and Cas13a variants for future Cas13-based RNA detection applications.
NUCLEIC ACIDS RESEARCH
(2023)