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SHERLOCK: nucleic acid detection with CRISPR nucleases. 2019

Max J Kellner, and Jeremy G Koob, and Jonathan S Gootenberg, and Omar O Abudayyeh, and Feng Zhang
Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.

UI MeSH Term Description Entries
D009696 Nucleic Acids High molecular weight polymers containing a mixture of purine and pyrimidine nucleotides chained together by ribose or deoxyribose linkages. Nucleic Acid,Acid, Nucleic,Acids, Nucleic
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D004720 Endonucleases Enzymes that catalyze the hydrolysis of the internal bonds and thereby the formation of polynucleotides or oligonucleotides from ribo- or deoxyribonucleotide chains. EC 3.1.-. Endonuclease
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000071243 Zika Virus Infection A viral disease transmitted by the bite of AEDES mosquitoes infected with ZIKA VIRUS. Its mild DENGUE-like symptoms include fever, rash, headaches and ARTHRALGIA. The viral infection during pregnancy, in rare cases, is associated with congenital brain and ocular abnormalities, called Congenital Zika Syndrome, including MICROCEPHALY and may also lead to GUILLAIN-BARRE SYNDROME. Congenital Zika Syndrome,Congenital Zika Virus Infection,Fever, Zika,ZikV Infection,Zika Fever,Zika Virus Disease,Disease, Zika Virus,Infection, ZikV,Infection, Zika Virus,Virus Disease, Zika,Virus Infection, Zika
D000071244 Zika Virus An arbovirus in the FLAVIVIRUS genus of the family FLAVIVIRIDAE. Originally isolated in the Zika Forest of UGANDA it has been introduced to Asia and the Americas. Virus, Zika,ZikV
D000094704 RNA, Guide, CRISPR-Cas Systems A component of CRISPR-Cas SYSTEMS. Cas endodeoxyribonucleases assemble with a guide RNA complex, a hybrid of CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) molecules, to form an active complex that cleaves DNA. crRNA and tracrRNA can be synthetically fused into a single RNA molecule, namely single guide RNA. Synthetic sgRNA is used with CRISPR-Cas SYSTEMS for targeted GENE EDITING. CRISPR Guide RNA,CRISPR-Cas Systems sgRNA (Single Guide RNA),Guide RNA (CRISPR-Cas Systems),Guide RNA, CRISPR-Cas Systems,RNA, CRISPR Guide,RNA, Guide (CRISPR-Cas Systems),RNA, Single Guide,RNA, Single-Guide,Single Guide RNA,Single-Guide RNA,Transactivating crRNA,crRNA,crRNA, Transactivating,sgRNA (CRISPR-Cas Systems),sgRNA (Single-Guide RNA),tracrRNA,Guide RNA, CRISPR,Guide RNA, CRISPR Cas Systems,Guide RNA, Single
D012260 Ribonucleases Enzymes that catalyze the hydrolysis of ester bonds within RNA. EC 3.1.-. Nucleases, RNA,RNase,Acid Ribonuclease,Alkaline Ribonuclease,Ribonuclease,RNA Nucleases,Ribonuclease, Acid,Ribonuclease, Alkaline
D015202 Protein Engineering Procedures by which protein structure and function are changed or created in vitro by altering existing or synthesizing new structural genes that direct the synthesis of proteins with sought-after properties. Such procedures may include the design of MOLECULAR MODELS of proteins using COMPUTER GRAPHICS or other molecular modeling techniques; site-specific mutagenesis (MUTAGENESIS, SITE-SPECIFIC) of existing genes; and DIRECTED MOLECULAR EVOLUTION techniques to create new genes. Genetic Engineering of Proteins,Genetic Engineering, Protein,Proteins, Genetic Engineering,Engineering, Protein,Engineering, Protein Genetic,Protein Genetic Engineering
D045183 Leptotrichia A genus of anaerobic, gram-negative bacteria in the family Fusobacteriaceae. Some species cause BACTEREMIA and some intra-amniotic infections.

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