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1.
Nat Commun ; 13(1): 1937, 2022 04 11.
Article in English | MEDLINE | ID: covidwho-1783981

ABSTRACT

In type II CRISPR systems, the guide RNA (gRNA) comprises a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA), both being essential in guided DNA targeting functions. Although tracrRNAs are diverse in sequence and structure across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for Cas9 is not fully understood. Here, we reveal the programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9, and in doing so, redefine the capabilities of Cas9 proteins and the sources of crRNAs, providing new biosensing applications for type II CRISPR systems. By reprogramming the crRNA-tracrRNA hybridized sequence, we show that engineered crRNA-tracrRNA interactions can not only enable the design of orthogonal cellular computing devices but also facilitate the hijacking of endogenous small RNAs/mRNAs as crRNAs. We subsequently describe how these re-engineered gRNA pairings can be implemented as RNA sensors, capable of monitoring the transcriptional activity of various environment-responsive genomic genes, or detecting SARS-CoV-2 RNA in vitro, as an Atypical gRNA-activated Transcription Halting Alarm (AGATHA) biosensor.


Subject(s)
Biosensing Techniques , COVID-19 , CRISPR-Cas Systems/genetics , Humans , RNA, Guide/genetics , RNA, Guide/metabolism , RNA, Viral/genetics , SARS-CoV-2/genetics
2.
Cell Rep ; 38(10): 110476, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1729612

ABSTRACT

Targeted delivery of therapeutic proteins toward specific cells and across cell membranes remains major challenges. Here, we develop protein-based delivery systems utilizing detoxified single-chain bacterial toxins such as diphtheria toxin (DT) and botulinum neurotoxin (BoNT)-like toxin, BoNT/X, as carriers. The system can deliver large protein cargoes including Cas13a, CasRx, Cas9, and Cre recombinase into cells in a receptor-dependent manner, although delivery of ribonucleoproteins containing guide RNAs is not successful. Delivery of Cas13a and CasRx, together with guide RNA expression, reduces mRNAs encoding GFP, SARS-CoV-2 fragments, and endogenous proteins PPIB, KRAS, and CXCR4 in multiple cell lines. Delivery of Cre recombinase modifies the reporter loci in cells. Delivery of Cas9, together with guide RNA expression, generates mutations at the targeted genomic sites in cell lines and induced pluripotent stem cell (iPSC)-derived human neurons. These findings establish modular delivery systems based on single-chain bacterial toxins for delivery of membrane-impermeable therapeutics into targeted cells.


Subject(s)
Bacterial Toxins , COVID-19 , Bacterial Toxins/genetics , CRISPR-Cas Systems , Gene Editing , Humans , RNA, Guide/metabolism , SARS-CoV-2
3.
PLoS One ; 16(12): e0261778, 2021.
Article in English | MEDLINE | ID: covidwho-1613357

ABSTRACT

Many CRISPR/Cas platforms have been established for the detection of SARS-CoV-2. But the detection platform of the variants of SARS-CoV-2 is scarce because its specificity is very challenging to achieve for those with only one or a few nucleotide(s) differences. Here, we report for the first time that chimeric crRNA could be critical in enhancing the specificity of CRISPR-Cas12a detecting of N501Y, which is shared by Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2 without compromising its sensitivity. This strategy could also be applied to detect other SARS-CoV-2 variants that differ only one or a few nucleotide(s) differences.


Subject(s)
COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , COVID-19/genetics , CRISPR-Cas Systems/genetics , DNA Primers/genetics , Diagnostic Tests, Routine/methods , Humans , Mutation/genetics , RNA, Guide/genetics , RNA, Guide/metabolism , Sensitivity and Specificity
4.
Chem Pharm Bull (Tokyo) ; 69(12): 1141-1159, 2021.
Article in English | MEDLINE | ID: covidwho-1546823

ABSTRACT

Considerable efforts have been made on the development of lipid nanoparticles (LNPs) for delivering of nucleic acids in LNP-based medicines, including a first-ever short interfering RNA (siRNA) medicine, Onpattro, and the mRNA vaccines against the coronavirus disease 2019 (COVID-19), which have been approved and are currently in use worldwide. The successful rational design of ionizable cationic lipids was a major breakthrough that dramatically increased delivery efficiency in this field. The LNPs would be expected to be useful as a platform technology for the delivery of various therapeutic modalities for genome editing and even for undiscovered therapeutic mechanisms. In this review, the current progress of my research, including the molecular design of pH-sensitive cationic lipids, their applications for various tissues and cell types, and for delivering various macromolecules, including siRNA, antisense oligonucleotide, mRNA, and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system will be described. Mechanistic studies regarding relationships between the physicochemical properties of LNPs, drug delivery, and biosafety are also summarized. Furthermore, current issues that need to be addressed for next generation drug delivery systems are discussed.


Subject(s)
Drug Carriers/chemistry , Lipids/chemistry , Liposomes/chemistry , Nanoparticles/chemistry , COVID-19/immunology , COVID-19/prevention & control , COVID-19/virology , Cations/chemistry , Hydrogen-Ion Concentration , RNA, Guide/chemistry , RNA, Guide/metabolism , RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , SARS-CoV-2/isolation & purification , /metabolism
5.
Chem Commun (Camb) ; 57(92): 12270-12272, 2021 Nov 19.
Article in English | MEDLINE | ID: covidwho-1506302

ABSTRACT

An automated Cas12a-microfluidic system was constructed to distinguish the B.1.617.2 (delta) variant of SARS-CoV-2 from the wild-type virus rapidly and was validated using 30 clinical samples, showing 100% consistency with next-generation sequencing. It will be a potential tool for the rapid differential diagnosis of the delta variant of SARS-CoV-2.


Subject(s)
COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Microfluidics/methods , SARS-CoV-2/genetics , Automation , COVID-19/virology , High-Throughput Nucleotide Sequencing , Humans , Mutation , Polymerase Chain Reaction , RNA, Guide/genetics , RNA, Guide/metabolism , RNA, Viral/metabolism , SARS-CoV-2/isolation & purification
6.
Cell ; 184(16): 4203-4219.e32, 2021 08 05.
Article in English | MEDLINE | ID: covidwho-1275187

ABSTRACT

SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques.


Subject(s)
Antibodies, Neutralizing/immunology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Viral/immunology , Bronchoalveolar Lavage Fluid/chemistry , COVID-19/pathology , COVID-19/virology , Cytokines/metabolism , Female , Haplorhini , Humans , Lung/pathology , Lung/virology , Male , Mice , Mice, Inbred BALB C , Protein Domains , RNA, Guide/metabolism , Receptors, IgG/metabolism , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Viral Load , Virus Replication
7.
Cell Rep Med ; 2(6): 100319, 2021 06 15.
Article in English | MEDLINE | ID: covidwho-1244849

ABSTRACT

There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/µL for a single guide RNA to 106 copies/µL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/µL and is completed using patient samples in less than 30 min.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , CRISPR-Cas Systems/genetics , RNA, Viral/metabolism , COVID-19/virology , Colorimetry , Humans , Molecular Diagnostic Techniques , Nasopharynx/virology , Nucleic Acid Amplification Techniques , RNA, Guide/metabolism , RNA, Viral/chemistry , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism
8.
Cell Rep Med ; 2(4): 100245, 2021 04 20.
Article in English | MEDLINE | ID: covidwho-1155662

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) and variants has led to significant mortality. We recently reported that an RNA-targeting CRISPR-Cas13 system, called prophylactic antiviral CRISPR in human cells (PAC-MAN), offered an antiviral strategy against SARS-CoV-2 and influenza A virus. Here, we expand in silico analysis to use PAC-MAN to target a broad spectrum of human- or livestock-infectious RNA viruses with high specificity, coverage, and predicted efficiency. Our analysis reveals that a minimal set of 14 CRISPR RNAs (crRNAs) is able to target >90% of human-infectious viruses across 10 RNA virus families. We predict that a set of 5 experimentally validated crRNAs can target new SARS-CoV-2 variant sequences with zero mismatches. We also build an online resource (crispr-pacman.stanford.edu) to support community use of CRISPR-Cas13 for broad-spectrum RNA virus targeting. Our work provides a new bioinformatic resource for using CRISPR-Cas13 to target diverse RNA viruses to facilitate the development of CRISPR-based antivirals.


Subject(s)
CRISPR-Cas Systems/genetics , RNA Viruses/genetics , RNA, Guide/metabolism , COVID-19/pathology , COVID-19/virology , Humans , RNA Virus Infections/pathology , RNA Virus Infections/virology , RNA Viruses/isolation & purification , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Species Specificity
9.
Cell Res ; 31(4): 395-403, 2021 04.
Article in English | MEDLINE | ID: covidwho-1091494

ABSTRACT

The upcoming flu season in the Northern Hemisphere merging with the current COVID-19 pandemic raises a potentially severe threat to public health. Through experimental coinfection with influenza A virus (IAV) and either pseudotyped or live SARS-CoV-2 virus, we found that IAV preinfection significantly promoted the infectivity of SARS-CoV-2 in a broad range of cell types. Remarkably, in vivo, increased SARS-CoV-2 viral load and more severe lung damage were observed in mice coinfected with IAV. Moreover, such enhancement of SARS-CoV-2 infectivity was not observed with several other respiratory viruses, likely due to a unique feature of IAV to elevate ACE2 expression. This study illustrates that IAV has a unique ability to aggravate SARS-CoV-2 infection, and thus, prevention of IAV infection is of great significance during the COVID-19 pandemic.


Subject(s)
COVID-19/pathology , Coinfection/pathology , Influenza A virus/physiology , Orthomyxoviridae Infections/pathology , SARS-CoV-2/physiology , Angiotensin-Converting Enzyme 2/deficiency , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/virology , Cathepsin L/genetics , Cathepsin L/metabolism , Cell Line , Coinfection/virology , Humans , Influenza A virus/isolation & purification , Lung/pathology , Mice , Mice, Transgenic , Orthomyxoviridae Infections/virology , RNA, Guide/metabolism , SARS-CoV-2/isolation & purification , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Severity of Illness Index , Viral Load , Virus Internalization
10.
Virus Res ; 294: 198282, 2021 03.
Article in English | MEDLINE | ID: covidwho-1019557

ABSTRACT

Type V and VI CRISPR enzymes are RNA-guided, DNA and RNA-targeting effectors that allow specific gene knockdown. Cas12 and Cas13 are CRISPR proteins that are efficient agents for diagnosis and combating single-stranded RNA (ssRNA) viruses. The programmability of these proteins paves the way for the detection and degradation of RNA viruses by targeting RNAs complementary to its CRISPR RNA (crRNA). Approximately two-thirds of viruses causing diseases contain ssRNA genomes. The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has caused the outbreak of the coronavirus disease 2019 (COVID-19), which has infected more than 88 million people worldwide with near 2 million deaths since December 2019. Thus, accurate and rapid diagnostic and therapeutic tools are essential for early detection and treatment of this widespread infectious disease. For us, the CRISPR based platforms seem to be a plausible new approach for an accurate detection and treatment of SARS-CoV-2. In this review, we talk about Cas12 and Cas13 CRISPR systems and their applications in diagnosis and treatment of RNA virus mediated diseases. In continue, the SARS-CoV-2 pathogenicity, and its conventional diagnostics and antivirals will be discussed. Moreover, we highlight novel CRISPR based diagnostic platforms and therapies for COVID-19. We also discuss the challenges of diagnostic CRISPR based platforms as well as clarifying the proposed solution for high efficient selective in vivo delivery of CRISPR components into SARS-CoV-2-infected cells.


Subject(s)
COVID-19/drug therapy , CRISPR-Cas Systems , SARS-CoV-2/drug effects , SARS-CoV-2/isolation & purification , Antiviral Agents/therapeutic use , COVID-19/diagnosis , COVID-19/therapy , COVID-19 Nucleic Acid Testing , CRISPR-Associated Proteins/therapeutic use , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Humans , RNA, Guide/genetics , RNA, Guide/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics
11.
CRISPR J ; 3(6): 487-502, 2020 12.
Article in English | MEDLINE | ID: covidwho-990516

ABSTRACT

Nucleic acid detection techniques are always critical to diagnosis, especially in the background of the present coronavirus disease 2019 pandemic. Simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, current nucleic acid detection techniques are still limited by traditional amplification and hybridization. To overcome this limitation, here we developed CRISPR-Cas9-assisted DNA detection (CADD). In this detection, a DNA sample is incubated with a pair of capture single guide RNAs (sgRNAs; sgRNAa and sgRNAb) specific to a target DNA, dCas9, a signal readout-related probe, and an oligo-coated solid support beads or microplate at room temperature (RT) for 15 min. During this incubation, the dCas9-sgRNA-DNA complex is formed and captured on solid support by the capture sequence of sgRNAa, and the signal readout-related probe is captured by the capture sequence of sgRNAb. Finally, the detection result is reported by a fluorescent or colorimetric signal readout. This detection was verified by detecting DNA of bacteria, cancer cells, and viruses. In particular, by designing a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), the HPV infection in 64 clinical cervical samples was successfully detected by the method. All detections can be finished in 30 min at RT. This detection holds promise for rapid on-the-spot detection or point-of-care testing.


Subject(s)
CRISPR-Associated Protein 9/metabolism , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Hybridization/methods , Animals , CRISPR-Cas Systems , DNA, Viral/genetics , Genetic Engineering/methods , Humans , Limit of Detection , Papillomavirus Infections/genetics , RNA, Guide/genetics , RNA, Guide/metabolism
12.
Angew Chem Int Ed Engl ; 59(46): 20545-20551, 2020 11 09.
Article in English | MEDLINE | ID: covidwho-966207

ABSTRACT

Modular construction of an autonomous and programmable multi-functional heterogeneous biochemical circuit that can identify, transform, translate, and amplify biological signals into physicochemical signals based on logic design principles can be a powerful means for the development of a variety of biotechnologies. To explore the conceptual validity, we design a CRISPR-array-mediated primer-exchange-reaction-based biochemical circuit cascade, which probes a specific biomolecular input, transform the input into a structurally accessible form for circuit wiring, translate the input information into an arbitrary sequence, and finally amplify the prescribed sequence through autonomous formation of a signaling concatemer. This upstream biochemical circuit is further wired with a downstream electrochemical interface, delivering an integrated bioanalytical platform. We program this platform to directly analyze the genome of SARS-CoV-2 in human cell lysate, demonstrating the capability and the utility of this unique integrated system.


Subject(s)
Biosensing Techniques/methods , Genes, Viral , SARS-CoV-2/genetics , COVID-19/pathology , COVID-19/virology , CRISPR-Cas Systems/genetics , Cell Line , Electrochemical Techniques , Humans , Nucleic Acid Amplification Techniques , RNA, Guide/metabolism , SARS-CoV-2/isolation & purification
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