CRISPR update

content/10.diagnostics.md

@@ -101,12 +101,12 @@ The SHERLOCK method (Specific High-sensitivity Enzymatic Reporter unLOCKing) fro
 The target RNA is amplified by RT-RPA and T7 transcription, and the amplified product activates Cas13a.
 The nuclease then cleaves a reporter RNA, which liberates a fluorescent dye from a quencher.
 Several groups have used the SHERLOCK method to detect SARS-CoV-2 viral RNA.
-An early study reported that the method could detect 7.5 copies of viral RNA in all 10 replicates, 2.5 copies in 6 out of 10, and 1.25 copies in 2 out of 10 runs [@doi:10.1101/2020.02.22.20025460].
+An early study reported that the method could detect 7.5 copies of viral RNA in all 10 replicates, 2.5 copies in 6 out of 10, and 1.25 copies in 2 out of 10 runs [@doi:10.1371/journal.ppat.1008705].
 It also reported 100% specificity and sensitivity on 114 RNA samples from clinical respiratory samples (61 suspected cases, among which 52 were confirmed and nine were ruled out by metagenomic next-generation sequencing, 17 nCoV-/HCoV+ cases and 36 samples from healthy subjects), and a reaction turnaround time of 40 minutes.
 A separate study screened four designs of SHERLOCK and extensively tested the best-performing assay.
 They determined the limit of detection to be 10 copies/μl using both fluorescent and lateral flow detection [@doi:10.1101/2020.02.26.967026].
 Lateral flow test strips are simple to use and read, but there are limitations in terms of availability and cost per test.
-Another group therefore proposed the CREST protocol (Cas13-based, Rugged, Equitable, Scalable Testing), which uses a P51 cardboard fluorescence visualizer, powered by a 9V battery, for the detection of Cas13 activity instead of immunochromatography [@doi:10.1101/2020.04.20.052159].
+Another group therefore proposed the CREST protocol (Cas13-based, Rugged, Equitable, Scalable Testing), which uses a P51 cardboard fluorescence visualizer, powered by a 9V battery, for the detection of Cas13 activity instead of immunochromatography [@doi:10.1128/JCM.02402-20].
 CREST can be run, from RNA sample to result, with no need for AC power or a dedicated facility, with minimal handling in approximately 2 hours.
 Testing was performed on 14 nasopharyngeal swabs.
 CREST picked up the same positives as the CDC-recommended TaqMan assay with the exception of one borderline sample that displayed low-quality RNA.
@@ -120,20 +120,37 @@ The assay had 95% positive predictive agreement and 100% negative predictive agr
 The estimated limit of detection was 10 copies per μl reaction, versus 1 copy per μl reaction for the CDC assay.
 These results have been confirmed by other DETECTR approaches.
 Using RTRPA for amplification, another group detected 10 copies of synthetic SARS-CoV-2 RNA per μl of input within 60 minutes of RNA sample preparation in a proof-of-principle evaluation [@doi:10.1101/2020.02.29.971127].
+Using a similar approach, another group reported detection at 1 copy per μl [@doi:10.1371/journal.pbio.3000978].
 The DETECTR protocol was improved by combining RT-RPA and CRISPR-based detection in a one-pot reaction that incubates at a single temperature, and by using dual crRNAs (which increases sensitivity).
-This new assay, known as All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR), detected 4.6 copies of SARS-CoV-2 RNA per μl of input in 40 minutes [@doi:10.1101/2020.03.19.998724].
-Another single-tube, constant-temperature approach using Cas12b instead of Cas12a achieved a detection limit of 5 copies/μl in 40-60 minutes [@doi:10.1101/2020.04.10.023358].
-It was also reported that electric field gradients can be used to control and accelerate CRISPR assays by co-focusing Cas12-gRNA, reporters, and target [@doi:10.1101/2020.05.21.109637].
+This new assay, known as All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR), detected 4.6 copies of SARS-CoV-2 RNA per μl of input in 40 minutes [@doi:10.1038/s41467-020-18575-6].
+Another single-tube, constant-temperature approach using Cas12b instead of Cas12a achieved a detection limit of 5 copies/μl in 40-60 minutes [@doi:10.1038/s41421-020-0174-y].
+It was also reported that electric field gradients can be used to control and accelerate CRISPR assays by co-focusing Cas12-gRNA, reporters, and target [@doi:10.1073/pnas.2010254117].
 The authors generated an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip.
 They also used ITP for automated purification of target RNA from raw nasopharyngeal swab samples.
 Combining this ITP purification with loop-mediated isothermal amplification, their ITP-enhanced assay to achieved detection of SARS-CoV-2 RNA (from raw sample to result) in 30 minutes.
 
+All these methods required upstream nucleic acid amplification prior to CRISPR-based detection. 
+They are relying on type V (Cas12-based) and type IV (Cas13-based) CRISPR systems.
+In contrast, type III CRISPR systems have the unique property of initiating a signalling cascade, which could boost the sensitivity of direct RNA detection. 
+A study tested this hypothesis using the type III-A CRISPR RNA (crRNA)-guided surveillance complex from Thermus thermophilus [@doi:10.1101/2020.10.14.20212670]. 
+They showed that activation of the Cas10 polymerase generates three products (cyclic nucleotides, protons, pyrophosphates) that can all be used to detect SARS-CoV-2 RNA. 
+Direct detection was 100-fold more sensitive than direct detection by Cas13. 
+Detection of viral RNA in patient samples still required an initial nucleic acid amplification step, but improvements may in the future remove that requirement.
+
+This goal of amplification-free detection was later achieved for a Cas13a-based system [@doi:10.1016/j.cell.2020.12.001]. 
+This approach combined multiple CRISPR RNAs to increase Cas13a activation, which is detected by a fluorescent reporter. 
+Importantly, because the viral RNA is detected directly, the test yields a quantitative measurement rather than a binary result. 
+The study also shows that fluorescence can be measured in a custom-made dark box with a mobile phone camera and a low-cost laser illumination and collection optics. 
+This makes this approach a truly portable assay for point-of-care diagnostics. 
+The authors achieved detection of 100 copies/μl of pre-isolated RNA in 30 minutes, and correctly identified all SARS-CoV-2-positive patient RNA samples tested in 5 minutes (n=20).
+
 There is an increasing body of evidence that CRISPR-based assays offer a practical solution for rapid, low-barrier testing in areas that are at greater risk of infection, such as airports and local community hospitals.
-In the largest study to date, DETECTR was compared to qRT-PCR on 378 patient samples [@doi:10.1101/2020.07.27.20147249].
+In the largest study to date, DETECTR was compared to qRT-PCR on 378 patient samples [@doi:10.1093/infdis/jiaa641].
 The authors reported a 95% reproducibility.
 Both techniques were equally sensitive in detecting SARS-CoV-2.
 Lateral flow strips showed a 100% correlation to the high-throughput DETECTR assay.
 Importantly, DETECTR was 100% specific for SARS-CoV-2 and did not detect other human coronaviruses.
+A method based on a Cas9 ortholog from Francisella novicida (FnCas9) achieved 100% sensitivity and 97% specificity in clinical samples, and the diagnostic kit is reported to have completed regulatory validation in India [@doi:10.1101/2020.09.13.20193581].
 
 #### Limitations of Molecular Tests