SHERLOCK is a CRISPR-based nucleic acid detection platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of DNA or RNA sequences. This platform allows for multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. The protocol provides 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 fluorescent and colorimetric readouts that provide results in less than 1 hour. Guidelines for designing efficient CRISPR RNA and isothermal amplification primers are also included, along with important considerations for multiplex and quantitative SHERLOCK detection assays. SHERLOCK uses Cas13 or Cas12 enzymes, which exhibit non-specific endonuclease activity after binding to a specific target via programmable CRISPR RNAs. By combining the programmable specificity of Cas13 or Cas12 with a reporter molecule that is activated upon target recognition, these enzymes result in specific and sensitive indication of the presence or quantity of a nucleic acid. The protocol describes a method for SHERLOCK nucleic acid detection using RPA and CRISPR-Cas13, including reagent preparation, recombinant Leptotrichia wadei Cas13 (LwaCas13a) protein expression and purification, as well as in vitro transcription of crRNAs and sample extraction from various starting materials. Cas13 is an RNA-guided RNase that produces multiple cleavage sites in single-stranded areas of an RNA target with specific base preferences. Cas13 also exhibits target-dependent promiscuous RNase activity, leading to trans cleavage of bystander RNA molecules, an effect termed "collateral activity." The discovery of collateral activity made it possible to develop a new approach for nucleic acid detection. Nucleic acid detection with collateral activity can be achieved using a variety of reporter molecules and methods, including fluorescent detection, fluorimeter detection, visual detection through modulation of solution turbidity, and lateral flow detection. SHERLOCK is highly sensitive and specific, capable of single molecule detection in 1uL sample volumes (2 aM) of both DNA and RNA. It can also detect single nucleotide mismatches in target sequences, allowing for the distinction of similar viruses such as Dengue and Zika. SHERLOCK can be used for the detection and genotyping of bacterial and viral infectious disease agents, including the detection of antibiotic resistance genes. It has been applied to detect Zika and Dengue virus directly from patient urine and serum samples, demonstrating the field-deployable nature of the technology. SHERLOCK is compatible with multiple readouts, either fluorescent detection or lateral flow detection, depending on the reporter molecule choice. While either Cas12 or Cas13 enzymes can be used for detection, thisSHERLOCK is a CRISPR-based nucleic acid detection platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of DNA or RNA sequences. This platform allows for multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. The protocol provides 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 fluorescent and colorimetric readouts that provide results in less than 1 hour. Guidelines for designing efficient CRISPR RNA and isothermal amplification primers are also included, along with important considerations for multiplex and quantitative SHERLOCK detection assays. SHERLOCK uses Cas13 or Cas12 enzymes, which exhibit non-specific endonuclease activity after binding to a specific target via programmable CRISPR RNAs. By combining the programmable specificity of Cas13 or Cas12 with a reporter molecule that is activated upon target recognition, these enzymes result in specific and sensitive indication of the presence or quantity of a nucleic acid. The protocol describes a method for SHERLOCK nucleic acid detection using RPA and CRISPR-Cas13, including reagent preparation, recombinant Leptotrichia wadei Cas13 (LwaCas13a) protein expression and purification, as well as in vitro transcription of crRNAs and sample extraction from various starting materials. Cas13 is an RNA-guided RNase that produces multiple cleavage sites in single-stranded areas of an RNA target with specific base preferences. Cas13 also exhibits target-dependent promiscuous RNase activity, leading to trans cleavage of bystander RNA molecules, an effect termed "collateral activity." The discovery of collateral activity made it possible to develop a new approach for nucleic acid detection. Nucleic acid detection with collateral activity can be achieved using a variety of reporter molecules and methods, including fluorescent detection, fluorimeter detection, visual detection through modulation of solution turbidity, and lateral flow detection. SHERLOCK is highly sensitive and specific, capable of single molecule detection in 1uL sample volumes (2 aM) of both DNA and RNA. It can also detect single nucleotide mismatches in target sequences, allowing for the distinction of similar viruses such as Dengue and Zika. SHERLOCK can be used for the detection and genotyping of bacterial and viral infectious disease agents, including the detection of antibiotic resistance genes. It has been applied to detect Zika and Dengue virus directly from patient urine and serum samples, demonstrating the field-deployable nature of the technology. SHERLOCK is compatible with multiple readouts, either fluorescent detection or lateral flow detection, depending on the reporter molecule choice. While either Cas12 or Cas13 enzymes can be used for detection, this