From 1a930187744904cbb19596854b79ed3e32ef5d01 Mon Sep 17 00:00:00 2001 From: Dimitri Perrin Date: Wed, 17 Feb 2021 22:30:58 +1000 Subject: [PATCH 1/2] CRISPR update * 6 pre-prints have now been published; citations have been updated * 1 new paper on an existing approach * 3 new approaches --- content/10.diagnostics.md | 29 +++++++++++++++++++++++------ 1 file changed, 23 insertions(+), 6 deletions(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 721c42290..9c5a8bbcf 100644 --- a/content/10.diagnostics.md +++ b/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 From 23afa98ac13321fb71d83d044c75b47a749f2f6e Mon Sep 17 00:00:00 2001 From: HM Rando Date: Thu, 6 Jan 2022 10:24:28 -0500 Subject: [PATCH 2/2] Update content/10.diagnostics.md Co-authored-by: nilswellhausen <62564517+nilswellhausen@users.noreply.github.com> --- content/10.diagnostics.md | 3 ++- 1 file changed, 2 insertions(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index a8a61660c..1b2fab265 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -148,7 +148,8 @@ Combining this ITP purification with loop-mediated isothermal amplification, the 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. +In contrast, type III CRISPR systems have the unique property of initiating a signaling 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.