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= CRISPR as a diagnostic tool = CRISPR and its brief origin

Significance

Diagnosis of disease- recent viral outbreaks especially during the 2015-16 Zika pandemic, where detection was difficult due to mild clinical symptoms. The diagnosis was a tricky aspect, as most of the symptoms lined up with Dengue virus (DENV). While contemporary nucleic acid detection methods are laborious and require time and equipment, the antigen-based assay lacks sensitivity. The use of CRISPR in this context could lead to a quick, sensitive, and universal diagnostic method. With tuberculosis, the sputum method is still employed and the turnaround time is about six to eight week in some cases. This period may be critical if the patient is emergent need for treatments. A low-cost diagnostic tool that is sensitive, specific is required.

History/Background
- Mechanism of CRISPR

-Current Diagnostic methods

Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK)
CRISPR associated proteins (Cas) are a part of the adaptive immune system discovered in bacteria. They have a “Lamarckians” mode of action that stores foreign DNA into host DNA to create a memory response via an RNA mediated process. This CRISPR mechanism has helped develop efficient and precise genome editing tools. An effector protein in the Class II system of CRISPR associated proteins discovered from Leptotrichia shahii, Cas13a (previously known as C2c2) is an RNA-guided nuclease that can be programmed with a guide RNA to target specific RNA sequences. This is a different mechanism of action compared to the famously known Cas9 protein, which targets DNA and causes a permanent change to the DNA. As RNA is expressed transiently, Cas13a can provide a window of targeting RNA without permanently altering the DNA. On the detection of target RNA, Cas13a cleaves all single stranded RNA (ssRNA) making it possible to detect a signal via cleavage of synthetic RNA that releases signaling molecule, this process is called 'collateral cleavage'.

Researchers at Broad Institute used Cas13a ortholog from Leptotrichia wadei due to its relatively high RNA guided RNAse activity compared to Leptotrichia shahii. To achieve attomolar (10-18 M) sensitivity, they combined an isothermal recombinase polymerase amplification step (RPA) to produced copies of target RNA quickly at a constant temperature. This was coupled with T7 mediated transcription to convert any DNA target sequence into RNA for detection if necessary. RPA eliminates the need to have a thermocycler which might not be accessible in remote point of care locations in foreign countries. RPA, T7 mediated transcription, and collateral cleavage following detection by Cas13a is termed SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing). Researchers demonstrated that SHERLOCK was able to detect and differentiate between Zika Virus (ZIKV) and Dengue virus (DENV) at an effective concentration of 2 aM. A paper-based assay developed was functional when the constituents were freeze-dried; they also demonstrated the ability to discriminate and target specific bacterial population using universal 16S rRNA gene V3 primers. Additionally, they were able to distinguish between two Klebsiella pneumoniae clinical isolates via antibiotic resistance genes. By introducing a single base mismatch in the crRNA, the researchers demonstrated highly specific strain SNP detection that in turn enabled to differentiate between American and African ZIKV. Since SNPs are a common element of human genome researchers proposed using SHERLOCK to perform rapid human genotyping and leveraging the high specificity they detected mutations in cell-free DNA fragments samples with specific allelic fractions present as low as 0.1%. This rapid detection was calculated to cost about $0.6/ reaction.

SHERLOCK v2.0
(i) four-channel single reaction multiplexed with other CRISPR orthologs

(ii) quantitative measurement as low as 2 attomolar

(iii) 3.5 fold increase in signal sensitivity by combining Cas13 with Csm6

(iv) lateral flow readout

Gootenberg et al. evaluated 14 members from the Cas13b family and 3 members from Cas13a family derived from various strains and species of bacteria. They evaluated the cleavage sequence preferences of these enzymes. They designed five nucleotide long homopolymer reporters and found that enzymes had varying preferences, but further evaluation using dinucleotides sequences pointed out four enzymes LwaCas13a, CcaCas13b, LbaCas13a, and PsmCas13b could be identified by AU, UC, AC, and GA dinucleotide sequences respectively. This specific cleavage preference enabled the detection of the ZIKV and DENV ssRNA simultaneously using unique nucleic acid sequence complexed with a fluorescent dye. Further coupling of enzymes possessing cleavage preference, with SHERLOCK mechanism, enabled detecting four targets in a sample using PsmCas13b, LwaCas13a, CcaCas13b, enzymes cleaving target ssRNA and AsCas12a cleaving ssDNA all linked to unique fluorophores. Unlike the previous iteration of SHERLOCK, SHERLOCK v2.0 demonstrated better quantitative measurements while using less primer for pre-amplification and mitigated the saturation of pre-amplification reaction that provided close correlation of input and signal intensity across a broad range of sample concentration. Another CRISPR nuclease, Csm6 was found to be activated by the cleavage activity of LwaCas13a and PsmCas13b, which generated a 2’3’ cyclic phosphate needed to activate the Csm6. Based on Csm6’ s preference for A and C rich reporters, the researchers designed RNA activators with poly-A stretch followed by a poly-U stretch that would first activate the LwaCas13a and then Csm6. This was confirmed by combining reporters for Csm6 and Cas13 in the same reaction conjugated with the same dye molecule. Increasing the activator increased the added activation of Csm6 by Cas13 for DENV ssRNA detection. Another improvement in this iteration was to develop a lateral flow paper-based readout method, that could be useful in making this diagnostic tool easily deployable in point of care centers and eliminated the need to have additional equipment for quantification.

Field-deployable viral diagnostics using CRISPR
The SHERLOCK mechanism shows unprecedented sensitivity and comprehensive ability to detect any ssRNA target as long as it is extracted. For SHERLOCK to be deployable, Cameron Myhrvold and colleagues from Broad Institute and Center for Systems Biology at Harvard University, proposed an extraction method that extracts viral nucleic acids directly from saliva and urine. HUDSON (heating unextracted diagnostic samples to obliterate nucleases) in concert with SHERLOCK was able to extract nucleic acids, inactivate ribonucleases, and detect DENV and ZIKV in 37 patients and 3 mosquito pools from 2015-16 pandemic. The results were compared with two FDA approved diagnostic tests for detecting ZIKV: Hologic Aptima ZIKV assay, and Altona Real Star Zika virus RT-PCR assay.

During each epidemic, diagnostic tests, surveillance, and bio-containment have been issues of concern. Scientists have used leveraged the ‘collateral activity’ of CRISPR associated enzymes to cleave ssRNA using Cas13 or ssDNA using Cas12a. Both the papers demonstrate the ease of conducting the test and using lyophilized reagents that confers the use in field operations by eliminating the need to cryopreserve the samples. Myhrvold and others were able to detect zika and dengue virus in patients with low concentrations, with the ability to distinguish dengue virus serotypes and subsequently identified region-specific Zika virus strain from 2015-16 pandemic. Not only restricted to infectious diseases, but researchers at UC Berkeley were able to use their detection technique called DETECTR to detect HPV 16 and 18, associated with cervical cancer with about 96% accuracy. This expands the realm of detection beyond infectious diseases and can be a gateway to the early detection of cancer. With all the advantages including quick turnaround time and easy to handle, this technology is restricted by the fact that it requires a specific RNA for detection. If the outbreak is sudden and the cause is unknown or if targeting viruses mutate rapidly like EBOV and ZIKV, in such cases deploying this diagnostic tool can be challenging.

= References =