Changing the production function of diagnostic tests

Next-generation diagnostics with CRISPR

Last week while reading Science I noticed a short and crucial article. Up to now CRISPR technology was focused on gene editing, now we can say that its usefulness is widening into diagnostics. It may change completely molecular diagnostics of "infectious diseases through detection of Zika virus (ZIKV), Dengue virus (DENV), and human papillomavirus (HPV) in human  samples, and noninfectious diseases, such as detection of gene mutations in circulating cell-free DNA from lung cancer patients." The production founction of lab testing would change completely.
Several articles explain details about it. The fight for patents is going to start again on CRISPR diagnostics. And this are unfortunately bad news.
Anyway, Science article reminds us:

These emerging diagnostic tools will by necessity be compared to standard diagnostics to ensure sensitivity and specificity and will need to be field-tested to guarantee performance in patient care settings, as environmental conditions and end-user application might affect performance. Proven assays, if affordable, promise to improve care in resource-limited settings where undifferentiated febrile illness is the norm and where gaps or delays in diagnosis, targeted care, and infection control contribute to infectious disease mortality and spread.

More details in The Verge.

CRISPR Diagnostics (for COVID-19)

CRISPR–Cas12-based detection of SARS-CoV-2

Applied technologies for detection of COVID are basically PCR molecular assays and immunoassays. However, CRISPR developments are entering into diagnostics and you may find the first example in Nature.

We report development of a rapid (<40 accurate="" and="" as12-based="" assay="" br="" crispr="" detection="" easy-to-implement="" extracts.="" flow="" for="" from="" lateral="" min="" of="" respiratory="" rna="" sars-cov-2="" swab="">We validated our method using contrived reference samples and clinical samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT–PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.

The role of CRISPR in diagnostics tests is going to increase.

Daido Moriyama 

Stop covid with CRISPR Diagnostics

With Crispr, a Possible Quick Test for the Coronavirus

Sherlock's quick, CRISPR-based coronavirus test gets emergency nod

STOP COVID

Point-of-care testing for COVID-19 using SHERLOCK diagnostics

Great!

The FDA granted its first emergency authorization for a CRISPR-based test for COVID-19, developed by Sherlock Biosciences, designed to turn results around in about an hour compared to the four to six hours needed for other molecular diagnostics.
The test is based on the company’s namesake technology, SHERLOCK, short for Specific High-sensitivity Enzymatic Reporter unLOCKing, a Cas13a-based CRISPR system that targets RNA rather than DNA. It looks for an RNA sequence specific to SARS-CoV-2, the virus that causes COVID-19, in patient samples taken from the upper airways with a swab or from airways in the lungs known as bronchoalveolar washing.
“If it’s there, it attaches to the Cas13 enzyme and activates it, which leads to the chewing up and cleaving of RNA probes,” Sherlock CEO Rahul Dhanda told FierceMedTech. When cleaved, those RNA molecules release a fluorescent signal to show the virus is present.

Stop Covid with CRISPR Diagnostics (2)

CRISPR–Cas12-based detection of SARS-CoV-2

Mammoth Biosciences (a firm founded by Jennifer Doudna) has partenered with GSK to commercialise a CRISPR Covid test. Therefore, there are right now two firms in the race: Sherlock and Mammoth.
The paper in Nature explains the details:

Here we report the development and initial validation of a CRISPR–Cas12-based assay9 for detection of SARS-CoV-2 from extracted patient sample RNA, called SARS-CoV-2 DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR). This assay performs simultaneous reverse transcription and isothermal amplification using loop-mediated amplification (RT–LAMP)14 for RNA extracted from nasopharyngeal or oropharyngeal swabs in universal transport medium (UTM), followed by Cas12 detection of predefined coronavirus sequences, after which cleavage of a reporter molecule confirms detection of the virus. We first designed primers targeting the E (envelope) and N (nucleoprotein) genes of SARS-CoV-2 (Fig. 1a). The primers amplify regions that overlap the World Health Organization (WHO) assay (E gene region) and US CDC assay (N2 region in the N gene)6,15, but are modified to meet design requirements for LAMP. We did not target the N1 and N3 regions used by the US CDC assay, as these regions lacked suitable protospacer adjacent motif sites for the Cas12 guide RNAs (gRNAs). Next, we designed Cas12 gRNAs to detect three SARS-like coronaviruses (SARS-CoV-2 (accession NC_045512), bat SARS-like coronavirus (bat-SL-CoVZC45, accession MG772933) and SARS-CoV (accession NC_004718)) in the E gene and specifically detect only SARS-CoV-2 in the N gene (Supplementary Fig. 1). This design is similar to those used by the WHO and US CDC assays, which use multiple amplicons with probes that are either specific to SARS-CoV-2 or are capable of identifying related SARS-like coronaviruses.

Edward Hopper 

Stop Covid with CRISPR Diagnostics (3)

 Detection of SARS-CoV-2 with SHERLOCK One-Pot Testing

Former posts have highlighted the potential of CRISPR for molecular  diagnostics, specially in case of Covid. Now NEJM provides details of Sherlock test.

Protocol here

Covid-19 testing landscape

COVID-19 diagnostics in context

This is the best summary of current supply of diagnostic tests for Covid-19:

COVID-19 tests can be grouped as nucleic acid, serological, antigen, and ancillary tests, all of which play distinct roles in hospital, point-of-care, or large-scale population testing.
Table 1 summarizes the existing and emerging tests, current at the time of writing (May 2020). A continuously updated version of this table is available at https://csb.mgh.harvard.edu/covid

Eric Topol says:

There are now *88* @US_FDA  cleared (by EUA) #COVID19 tests so far. Their false negative rates range from 10-48% (by post-release reports).
Might be better to have less tests, more accuracy, with faster turnaround

I agree.

Table 1 Performance comparison of different test types.
Throughput is determined by process type and assay time. In general, automated plate-based assays have higher daily throughputs. Hashtag (#) indicates example systems that have received FDA emergency use authorization (FDA-EUA). See https://csb.mgh.harvard.edu/covid to access continuously updated information. PCR, polymerase chain reaction; PCR-POC, PCR–point-of-care; ddPCR, digital droplet PCR; NEAR, nicking endonuclease amplification reaction; RCA, rolling circle amplification; SHERLOCK, specific high-sensitivity enzymatic reporter; DETECTR, DNA endonuclease-targeted CRISPR transreporter; NGS, next-generation sequencing; μNMR, micro–nuclear magnetic resonance; LFA, lateral flow assay; ELISA, enzyme-linked immunosorbent assay; CLIA, chemiluminescence immunoassay; EIA, enzyme immunoassay; ECLIA, electrochemiluminescence immunoassay; ECS, electrochemical sensing; VAT, viral antigen assay; IFM, immunofluorescence microscopy; WB, Western blot.

Type	Target	Virus	Assay time	Process type	FDA-EUA	Examples
PCR	Viral RNA	SARS-CoV-2	2–8 hours; >12 hours	Plate	56	#Roche, #LabCorp, #BioMerieux, #Qiagen, #Perkin-Elmer, #Becton Dickinson, #Luminex, #Thermo Fisher, others
PCR-POC	Viral RNA	SARS-CoV-2	<1 hour="" td="">	Cartridge	2	#Cepheid, #Mesa, Credo
ddPCR	Viral RNA	SARS-CoV-2	2–4 hours	Manual	1	#BioRAD
NEAR	Viral RNA	SARS-CoV-2	15 min	Cartridge	1	#Abbott
OMEGA	Viral RNA	SARS-CoV-2	1 hour	Plate	1	#Atila BioSystems
RCA	Viral RNA	SARS-CoV	2 hours		0
SHERLOCK	Viral RNA	SARS-CoV-2	1.5 hours	Kit	1	#Sherlock Biosciences (CAS13a)
DETECTR	Viral RNA	SARS-CoV-2	1 hour	Kit	0	Mammoth Biosciences (CAS12a)
NGS	Viral RNA	SARS-CoV-2	Days		1	#IDbyDNA, Vision, Illumina
μNMR	Viral RNA	SARS-CoV-2	2 hours	Cartridge	0	T2 Biosystems
LFA	IgG, IgM	SARS-CoV-2	15 min	Cartridge	3	#Cellex, #Sugentech, #ChemBio, Innovita
ELISA	IgG, IgM	SARS-CoV-2	2–4 hours	Plate	4	#Mount Sinai, #Ortho-Clinical (2), #EUROIMMUN US Inc., BioRAD, Snibe, Zhejiang orient, Creative Dx
CLIA	IgG, IgM	SARS-CoV-2	30 min	Cartridge	2	#Abbott, #DiaSorin
EIA	IgG, IgM	SARS-CoV-2	2 hours	Plate	1	#BioRAD
MIA	IgG, IgM	SARS-CoV-2		Plate	1	#Wadsworth Center
ECLIA	IgG, IgM	SARS-CoV-2	20 min	Plate	1	#Roche
ECS	IgG, cytokine	SARS-CoV-2	1 hour	Cartridge	0	Accure Health
VAT	Viral antigen	SARS-CoV-2	20 min	Cartridge	1	#Quidel, Sona NT, RayBiotech, SD Biosensors, Bioeasy
Microarrays	Ig epitopes	SARS-CoV-2	1.5 hours	Plate	0	RayBiotech, PEPperPRINT
IFM	Viral protein	SARS-CoV	3 hours	Manual	0
WB	IgG, IgM; viral protein	SARS-CoV	4 hours	Manual	0