SHERLOCK won't miss a single mutation
Anna Kaznadzei, Alexander Ershov, N+1
Fast, inexpensive and highly accurate detection of nucleic acids will now be possible using the SHERLOCK method based on the operation of the CRISPR-Cas13a system. It can be used to identify pathogens, genotyping and monitoring of genetic diseases. Article by Gootenberg et al. Nucleic acid detection with CRISPR-Cas13a/C2c2 is published in Science.
The key element of the new technology is the Cas13a nuclease, formerly known as C2c2, an enzyme that is able to recognize a specific sequence in an RNA strand and then cut any RNAs that come close to it. We have already written about the discovery of Cas13a/C2c2 earlier. In its function, this nuclease is similar to the more well-known Cas9, which is widely used for genome editing. However, unlike the latter, Cas13a/C2c2 has two important differences: firstly, it works at the level of RNA, not DNA, and secondly, it does not introduce a "surgical precise" gap into the target, but completely destroys it. Such features make it unsuitable for genome editing, however, as it turned out, they can also be successfully used for completely different purposes.
The nucleic acid detection system developed on the basis of Cas13a/C2c2 makes it possible to work with attomolar (10-18 mol per liter) concentrations of DNA or RNA, and at the same time quickly and accurately detect different strains of viruses, distinguish pathogenic bacteria, genotype human DNA and determine tumor mutations in extracellular DNA circulating in blood plasma.
The basic scheme of the SHERLOCK method: all nucleic acid from the sample is converted into DNA, RNA is synthesized on its basis using T7 polymerase, and then samples of the desired sequence are found in this "library" using Cas13a/C2c2 nuclease armed with a "reference sample" of this sequence. The destruction of RNA leads to fluorescence. Since Cas13a/C2c2 is a process, it does not stop until it destroys all the molecules that match the sequence (figure from the article in Science).
In order to investigate the capabilities of this system, the Cas13a protein of the bacterium Leptotrichia shahii was incubated with target RNA (ssRNA 1), CRISPR RNA (crRNA) and reporter RNA, which made it possible to make sure that the enzyme worked. At the same time, fragments of ssRNA 1 were encoded in the CRISPR cassette, since it is the enzyme that uses it as a cheat sheet for searching for the target RNA. The system worked in vitro and showed good sensitivity, but in order to achieve the attomolar sensitivity of the experiment, scientists had to select the conditions for pre-amplification (a method of increasing the number of nucleic acids) for some time. Optimal enzyme performance was achieved by recombinase polymerase amplification, which increases the number of DNA copies, after which DNA was converted into RNA, after which ssRNA 1 was detected using Cas13a. The enzyme sensed a certain sequence of ssRNA 1, comparing it with the one encoded in CRISPR, and cut the surrounding RNA, including the reporter one. The cut reporter RNA began to fluoresce, making it possible to make sure that the enzyme had found the target. This set of methods was called SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing).
Drip-digital PCR methods have confirmed that only one molecule of the original RNA or DNA is enough for this system to work. The sensitivity of SHERLOCK coincides with the sensitivity of the quantitative PCR method and drip-digital PCR – the two main methods of detecting nucleic acids, while its results with multiple repetitions of the experiment show less variability.
The effectiveness of the method was tested on lentiviruses containing genomic fragments of Zika virus and Dengue virus. SHERLOCK recognized viral particles whose concentration did not exceed two attomoles, and distinguished the two types mentioned above from each other. It also turned out that SHERLOCK can be subjected to drying and subsequent restoration, and its sensitivity does not fall below 20 attomol. Detection in this case can be carried out on fiberglass paper, which makes the method convenient for use in almost any conditions.
The action of SHERLOCK was tested on urine, saliva and serum samples, where it successfully detected nucleic acids with a concentration of about three attomoles. At the same time, the nucleic acids did not have to be pre-purified. SHERLOCK also correctly distinguishes between different strains of pathogenic bacteria. Using mutagenesis methods to introduce artificial mutations, scientists have found out that the system allows detecting even single nucleotide substitutions (SNPs). This, in turn, makes it possible to carry out genotyping of human genomes, which is based on their single-nucleotide differences. SHERLOCK successfully "identified" four human samples from five genomic loci taken from the 23andMe database, and could detect both homozygous and heterozygous mutations. The last stage was the search for extracellular DNA fragments with cancer mutations floating freely in blood samples of a sick person, the detection of which is usually difficult due to the large amount of ordinary DNA. It turned out that the method is able to record even a 0.1 percent concentration of nucleic acid carrying cancer mutations.
Scientists believe that the use of SHERLOCK can become an important step in the methods of quantitative assessment of nucleic acids, multiplex expression assays and other sensitive techniques, including determining the level of contamination of samples. It can also be used to detect pathogenic mutations in living cells and allele-specific expression. One test on fiberglass paper will cost only about 61 cents.
Details about the prospects of CRISPR research and the history of the discovery of Cas13a/C2c2 can be found in a recent interview with one of the authors of this discovery, Konstantin Severinov.
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14.04.2017