Created by: Leena Dhaliwal, Adnan Hassanali, Tristen Nimojan, Abdullah Syeddan

Figure 1: Illustration of SARS-CoV-2. Adapted from Eckert & Higgins (2020).

Within the last 40 years, there have been many large-scale epidemics from viruses such as HIV, SARS and Middle East respiratory syndrome coronaviruses, 2009 pandemic influenza H1N1 virus, Ebola virus, Zika virus, and most recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Broughton et al., 2020). Like all the other epidemics, the pandemic resulting from SARS-CoV-2 is believed to be of zoonotic origin which spread into vulnerable human populations following an animal to human transmission event (Broughton et al., 2020). In December 2019 there was a disease outbreak caused by this virus, originating in China, which quickly spread across the globe and was declared a global pandemic on March 11, 2020 by the World Health Organization (Broughton et al., 2020). The name of the disease caused by this virus is coronavirus disease 2019, more commonly referred to as COVID-19 (Broughton et al., 2020).

During each of these epidemics, the lack of access to rapid and accurate diagnostic testing hindered the public health response to the virus (Broughton et al., 2020). Diagnostics have become especially important in this recent pandemic as many reported person-to-person transmission events have resulted from individuals who presented mild symptoms or were completely asymptomatic (Broughton et al., 2020). These methods of transmission greatly increase the number of individuals that need to be screened (Broughton et al., 2020). Several laboratories have developed diagnostic assays using quantitative reverse transcription polymerase chain reaction (qRT-PCR) however, there are some limitations to these assays (Broughton et al., 2020). For example, the assay developed by the Centers for Disease Control and Prevention (CDC) (2020) takes four to six hours to detect the virus and more than twenty-four hours for the overall turnaround time for screening and diagnosing patients. Additionally, the assay requires the use of expensive equipment such as the PRC Workstation and the qRT-PCR Detection System and requires higher expertise and technical skills to operate and interpret the results (CDC, 2020). Because of this expensive equipment and the need for trained personnel, the use of this assay is limited to public health laboratories who have access to these resources (Broughton et al., 2020). Similarly, serological tests may have limitations when it comes to diagnosing SARS-CoV-2 (Broughton et al., 2020). Although serological tests are rapid, these assays are limited by the patient’s immune response (Broughton et al., 2020). It may take several days to weeks for a patient to mount an antibody response at a level that can be detected by the assay (Broughton et al., 2020). This wait-time would hinder the diagnostic testing process and thus cause a delay in treatment and isolation procedures.

The Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-associated protein 12 or CRISPR-Cas12 based assay used for the detection of SARS-CoV-2 from purified patient sample RNA, developed by Broughton et al. (2020), circumvents some of these issues proposed by these previously developed assays. The assay is called SARS-CoV-2 DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) and it can detect SARS-CoV-2 in clinical samples within thirty to forty minutes (Broughton et al., 2020). Compared to qRT-PCR, DETECTR uses routine protocols and commercially available reagents while maintaining a comparable accuracy to qRT-PCR (Broughton et al., 2020). Some key advantages of this system compared to the CDC assay and other assays using qRT-PCR is the use of isothermal signal amplification which removes the need for thermocycling, rapid turnaround time, single nucleotide target specificity, the use of easy-to-use reporting formats such as lateral flow strips, and the non-requirement of a complex laboratory infrastructure (Broughton et al., 2020).

This CRISPR/Cas technology is not novel to the diagnosing of SARS-CoV-2 in 2020 but has been in development for the past few years now and several diagnostic kits have been developed and validated using Cas9, Cas12, Cas13 proteins (Srivastava et al., 2020). CRISPR/Cas technology involves making a specific guide RNA to recognize a sequence of interest and attaching it to the endonuclease enzyme, Cas (Srivastava et al., 2020). The guide RNA is made up of two components: CRISPR RNA, which is a nucleotide sequence complementary to the target DNA, and a tracr RNA, which serves as a binding scaffold for the Cas nuclease (Srivastava et al., 2020). Cas uses this guide RNA to locate, bind, and subsequently cleave the target DNA sequence within the sample using its endonuclease activity (Srivastava et al., 2020). The Cas protein does not cleave randomly within the target sequence, but it cleaves the segment of the sequence that is followed by a protospacer adjacent motif (PAM). PAM sequences typically consist of two to six nucleotide base pairs and are specific to the particular Cas nuclease (Srivastava et al., 2020).

To begin, the first use of CRISPR/Cas 9 technology in the diagnostics of viral infections was in 2016 during the Zika virus outbreak (Pardee et al., 2016). The name of the platform they used was called nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique based on CRISPR cleavage (NASBACC) (Pardee et al., 2016). Following the development of this assay, other diagnostics were developed using Cas9 (Srivastava et al., 2020). However, it was not until the discovery of other Cas proteins that could be used to create new assays, namely Cas12 and Cas13, that this technology gained traction around the world (Srivastava et al., 2020). In 2017, the molecular detection platform Specific High-Sensitivity Enzymatic Reporter Unlocking (SHERLOCK) was developed using Cas13 to detect specific strains of Zika and Dengue virus (Gootenberg et al., 2017). This platform was also used in other applications such as the differentiation of pathogenic bacteria, genotyping human DNA, and identifying cell-free tumor DNA mutations (Gootenberg et al., 2017). This assay was a step up from NASBACC (Pardee et al., 2016) as it was able to detect nucleic acids while maintaining high sensitivity and single-base specificity which was not maintained to the same extent in the 2016 assay (Gootenberg et al., 2017). Next, in 2018, a CRISPR/Cas12-based diagnostic system named one-HOur Low-cost Multipurpose highly Efficient System (HOLMES) was developed, however no specific disease was tested, rather premade DNA strands were utilized for validation (Li et al., 2018). Later in 2018, the SHERLOCK assay was upgraded to include multiplexing which is the use of multiple guide RNAs or Cas enzymes (Gootenberg et al., 2018). With the upgrades made to this assay, namely the use of both Cas13 and Cas12a, it was able to achieve high quantification and increased sensitivity (Gootenberg et al., 2018). Preceding the assay developed by Broughton et al. (2020) was the All-in-One Dual CRISPR (AIOD-CRISPR) assay which made use of Cas12a and aimed to detect SARS-CoV-2 and HIV virus (Ding et al., 2020). Unlike AIOD-CRISPR, the DETECTR assay focused on SARS-CoV-2 detection and made use of multiplexing (of guide RNAs) which functioned to increase the scope and efficiencies of this technology (Broughton et al., 2020).

James P Broughton and Xianding Deng, the lead authors of the paper “CRISPR-Cas12-based detection of SARS-CoV-2,” which developed the DETECTR assay, are part of Mammoth Biosciences, Inc. and the Department of Laboratory Medicine at the University of California San Francisco (UCSF) respectively (Broughton et al., 2020). Broughton is the senior manager at Mammoth Biosciences and has been in twelve publications including this paper from 2011 to 2020. Deng has been in twenty-four publications, including this paper and has 686 citations (ResearchGate, n.d.). His main research interest is using next generation sequencing, namely Illumina and nanopore, for investigating infectious diseases in the context of diagnostics, outbreak response, genomic sequencing, and antimicrobial surveillance (ResearchGate, n.d.). The remainder of the contributing authors are also affiliated with this company and university department in addition to other departments associated with USCF such as USCF-Abbott Viral Diagnostics and Discovery Center, and the Department of Biochemistry and Biophysics (Broughton et al., 2020). Furthermore, some of the authors are members of the Viral and Rickettsial Disease Laboratory within the California Department of Public Health (Broughton et al., 2020).

Mammoth Biosciences, Inc. is a biotechnology company which aims to develop easy-to-use, affordable point-of-care tests that use CRISPR technology (Mammoth Biosciences, Inc., 2020). With their exclusive licensing to Cas12, Cas13, and Cas14, they are working toward building the platform for CRISPR applications of the future not only in the context of diagnostics, but also to address other challenges across healthcare, agriculture, environmental monitoring, biodefense, and more (Mammoth Biosciences, Inc., 2020). Mammoth Biosciences deals with diagnostics, genome editing, and protein discovery to explore and solve these challenges (Mammoth Biosciences, Inc., 2020).

The University of California, San Francisco (UCSF) is known to be one of the world’s leading health science universities and is a major center of medical and biological research and education (UCSF, 2021). The university has produced many accomplished clinicians and scientists over the years (UCSF, 2021). The Department of Medicine is the largest department within UCSF School of Medicine and is one of the top four departments of medicine within the United States (UCSF, 2021). The department has published more than 2,491 peer reviewed articles during 2018 to 2019 alone and has since published more, covering various topics in health care and medicine such as COVID-19 (UCSF, 2021).

The Viral and Rickettsial Disease Laboratory (VRDL) within the California Department of Public Health, established in 1939, was the first state public health virology laboratory in the United States (California Department of Public Health, 2020). The aim of this laboratory is to provide high quality laboratory services and testing for viral pathogens that are significant to public health (California Department of Public Health, 2020). VRDL includes the following sections: The Vaccine Preventable Diseases and Herpes Viruses Section, The Respiratory and Gastroenteritis Diseases Section, The Zoonotic and VectorBorne Diseases Section, The Molecular Immunoserology Section, and The Data, Testing, Epidemiology, and Quality Support Section (California Department of Public Health, 2020). The laboratory has worked on the novel coronavirus, acute flaccid myelitis, measles and mumps, hepatitis A, West Nile and St, Louis encephalitis, and influenza (California Department of Public Health, 2020).

The SARS-CoV-2 DETECTR assay begins with simultaneous nucleic acid amplification and reverse transcription of RNA extracted from nasopharyngeal or oropharyngeal swabs in universal transport medium (Broughton et al., 2020). The amplified sample then undergoes Cas12-based detection of coronavirus-specific sequences followed by the subsequent cleavage of reporter molecules upon detection of the virus (Broughton et al., 2020). Visualization of the assay’s results can occur using fluorescent readers or lateral flow strips (Broughton et al., 2020). The assay’s primer targets include the envelope (E) and nucleoprotein (N) genes of SARS-CoV-2 modified to fit the RT-LAMP assay as per the recommendation of the WHO and the US CDC (Broughton et al., 2020). The N1 and N3 regions used by the US CDC are excluded from this assay due to a lack of PAM sites for the Cas12 gRNA (Broughton et al., 2020). The Cas12 gRNAs have been designed to detect the SARS-COV-2, bat-SL-CoVZC45 and SARS-CoV envelope genes and the SARS-CoV-2 nucleoprotein gene alone (Broughton et al., 2020). In conclusion, conventional RNA extraction methods are used as an input to DETECTR (RT-LAMP amplification and Cas12-based detection of E and N genes) which generate results visualized via fluorescent readers or lateral flow strips (Broughton et al., 2020).

SARS-CoV-2 target sequences were designed using genomes available from GISAID and viral genomes were aligned using Clustal Omega (Broughton et al., 2020). The LbCas12a SARS-CoV-2 target sites were filtered against SARS-CoV, two bat SARS-like CoV and other human coronavirus genomes with compatible sites compared to those used by the CDC and WHO (Broughton et al., 2020). LAMP primers were designed against SARS-CoV-2 N and E genes with compatible gRNAs (Broughton et al., 2020). Producing the target RNA involved performing PCR on the synthetic gene fragments of the target followed by an IVT reaction and heat-digestion (Brouhgton et al., 2020). The RNA was then purified and quantified in nuclease-free water to working concentrations (Broughton et al., 2020).

DETECTR assays were performed using RT-LAMP for amplification of viral targets and LbCas12a for the cleavage assay (Broughton et al., 2020). RT-LAMP reactions were performed independently for the N and E genes (Broughton et al., 2020). After the LbCas12a cleavage assay a lateral flow strip was added to the reaction tube where results showing a single band or double band close to the top of the strip indicated a positive result (Broughton et al., 2020).

Patient-optimized DETECTR methods were performed using RT-LAMP with a DNA-binding dye SYTO9 used to monitor the amplification step and incubation periods extended to thirty minutes (Broughton et al., 2020). Patient-optimized fluorescence-based LbCas12a cleavage assays were performed with preincubated LbCas12a-gRNA and a fluorescent reporter molecule compatible with the SYTO9 dye (Broughton et al., 2020). These modifications were made to capture data from \lower titer samples like patient-derived samples (Broughton et al., 2020).

Negative and positive nasopharyngeal and oropharyngeal swabs were acquired from the Chiu laboratory and UTM (Broughton et al., 2020). Samples of SARS-CoV-2 RNA were extracted using standard CDC EUA protocol (Broughton et al., 2020).

Fig. 1: This graph shows the gRNA specificity of the DETECTR system. The N gene gRNA on the left of the graph is specific for SARS-CoV-2, whereas the E gene gRNA on the right is able to detect three SARS-like coronavirus strains (right). Another N gene gRNA (shown in the middle) was designed to target SARS-CoV and a bat coronavirus but failed to detect SARS-CoV-2 (Broughton et al. 2020).

The DETECTR assay performs simultaneous reverse transcription and isothermal amplification using loop-mediated amplification (RT–LAMP) (Notomi et al. 2000) for RNA that can be extracted from either nasopharyngeal or oropharyngeal samples. Then Cas12 detection of predefined coronavirus sequences occurs where a reporter molecule is cleaved to confirm the detection of SARS-CoV2 (Broughton et al. 2020). Primers are designed to target the envelope (E) and nucleoprotein (N) genes of SARS-CoV-2 (Fig. 1) while still meeting the design requirements for LAMP. The N1 and N3 regions used by the US CDC assay were not used since these regions lacked suitable PAM (protospacer adjacent motif) sites for Cas12 guide RNAs (gRNAs) (Broughton et al. 2020).

Cas12 gRNAs are programmed to either specifically target SARS-CoV-2 or broadly detect related coronavirus strains as shown in Fig 1. Cas12 gRNAs were designed to detect three SARS-like coronaviruses (SARS-CoV-2, bat SARS-like coronavirus and SARS-CoV) in the E gene and, while only detecting SARS-CoV-2 in the N gene (Broughton et al. 2020). Using synthetic, in vitro-transcribed SARS-CoV-2 RNA gene targets, CRISPR–Cas12-based detection was able to distinguish SARS-CoV-2 specifically with no cross-reactivity for other coronavirus strains using N gene gRNA and with expected cross-reactivity for E gene gRNA (Broughton et al. 2020).

Optimized conditions for the SARS-CoV-2 DETECTR assay on the E gene, N gene and human RNase P gene as a control, consists of an RT–LAMP reaction at 62 °C for 20–30 min and Cas12 detection reaction at 37 °C for 10 min (Broughton et al. 2020).

Fig 2: Representation of a DETECTR system lateral flow readout. Intact FAM-biotin reporter molecule flows to the control capture line. When the Cas12 system finds its target, the Cas–gRNA complex cleaves the reporter molecule, which flows to the target capture line (Broughton et al. 2020).

The DETECTR assay is able to run in approximately 30–40 min and visualized on a lateral flow strip. The SARS-CoV-2 DETECTR assay is considered positive if there is detection of both the E and N genes or presumptive positive if there is detection of either the E or N gene (Fig. 3) (Broughton et al. 2020). Visualization of the Cas12 detection reaction is achieved using a FAM-biotin reporter molecule and lateral flow strips designed to capture the reporter molecules in different states (Myhrvold et al. 2018). Uncleaved reporter molecules are captured at the first detection line which is used as the “control line”. The second detection line is the “test line” where the reporter molecules cleaved by Cas12 activity create a signal (Fig. 2) (Broughton et al. 2020).

To compare the signal generated by Cas12 when using either fluorescence or lateral flow, RT–LAMP using 5-fM or 0-fM in vitro-transcribed (IVT) template was performed using N gene primers and the time to achieve a readout on identical amplicons was monitored by fluorescent plate reader and lateral flow (Broughton et al. 2020). The fluorescence signal of the LbCas12a reaction on the on RT–LAMP amplicon detection assay for the SARS-CoV-2 N gene completely saturates on a lateral flow strip within 10 min but can be seen within 5 min. The Cas12 fluorescent signal was detectable in less than 1 min and a visual signal by lateral flow was achieved within 5 min (Fig. 4) (Broughton et al. 2020).

Fig 4: Comparison of fluorescence readouts to lateral flow readouts using the DETECTR assay. Fluorescence of the LbCas12a detection assay for the SARS-CoV-2 N gene produces a signal almost immediately and saturates within 10 min, while the lateral flow readout produces a visible signal within 5 min (Broughton et al. 2020).

Fig 3: List of lateral flow strip assay readout results. A positive result requires detection of at least one of the two SARS-CoV-2 viral gene targets (N gene or E gene) (QC = Quality Control) (Broughton et al. 2020).

The analytic limit of detection (LoD) of the DETECTR fluorescent assay was compared to the US FDA EUA-approved CDC assay for detection of SARS-CoV-2 (Fig. 5) (Broughton et al. 2020). A standard curve for quantitation was constructed using seven dilutions of a control IVT viral nucleoprotein RNA, with three replicates at each dilution. Ten twofold serial dilutions of the same control nucleoprotein RNA were then used to run the fluorescent DETECTR assay, with six replicates at each dilution (Broughton et al. 2020). This was used to confirm that the assay can generate a visual signal by lateral flow at the LoD. The estimated LoD for the CDC assay is 1 copy per µl reaction, versus 10 copies per µl reaction found for the DETECTR assay.

Then the capability of the RT–LAMP assay to amplify SARS-CoV-2 nucleic acid directly from the raw samples from asymptomatic donors was assessed by placing the samples in buffer solutions such as UTM or phosphate-buffered saline, and then spiking these buffers with SARS-CoV-2 IVT target RNA (Broughton et al. 2020). The assay performance was diminished at reaction concentrations of ≥10% UTM and ≥10% phosphate-buffered saline by volume, with estimated LoDs decreasing to 15,000 and 500 copies per µl, respectively (Broughton et al. 2020).

Fig 5: Limit of detection for the CDC qPCR and DETECTR assays. These graphs show the serial dilution of viral copies in a sample and both assays ability to generate a signal at these diluted concentrations. The lateral flow strips show the results of the DETECTR assay shown at 0 copies per µl and 10 copies per µl (Broughton et al. 2020).

To test cross reactivity, RNA was extracted from 11 samples from six PCR-positive COVID-19 patients and 12 samples from patients with influenza and common human seasonal coronavirus infections using the SARS-CoV-2 DETECTR assay with fluorescence-based and lateral flow strip (Broughton et al., 2020). SARS-CoV-2 was detected in 9 of 11 patient swabs where two negative swabs from COVID-19 patients were confirmed to be below the established limit of detection. This test showed that there was no cross-reactivity with other respiratory viruses.

When compared, the lateral flow and fluorescence-based showed the same readout 95.8% of the time (Broughton et al., 2020). 60 samples from patients with acute respiratory infection were tested using a fluorescence-based readout to blindly test for SARS-CoV-2 using the DETECTR assay. 30 of these samples were positive for COVID-19 infection by qRT–PCR testing and 30 were negative for COVID-19 infection where these 30 samples were either positive for another viral respiratory infection when using multiplex PCR testing or negative by all testing (Broughton et al., 2020). The positive predictive agreement and negative predictive agreement of SARS-CoV-2 DETECTR compared to the CDC qRT–PCR assay were 95% and 100%, respectively, for detection of the coronavirus in a test of 83 total samples.

This technology has been created in order to be used at point of care. A sample and the test can be directly performed without the need to transfer the samples to a testing facility or the use of big machinery and specialized equipment (Broughton et al., 2020). Additionally, with easy read out of testing strips, training and special technicians are not required (Broughton et al., 2020). Furthermore, with lack of specialized machinery and transport of samples, the technique is more cost effective while still using affordable reagents (Broughton et al., 2020). The greatest benefit is being able to get results quickly, within 40 mins as compared to the long wait time of getting RT-PCR results (Broughton et al., 2020). While validation has showcased a 95% positive predictive agreement and 100% negative predictive agreement, clinical validation must commence under the FDA approved clinically licensed laboratories and approval by EUA (Broughton et al., 2020).

The use of Cas12 proteins relies on protospacer adjacent motif (PAM) sequences which is required for endonuclease activity (MammothBioscience, 2019). Therefore, targets are limited to areas in the genome where a PAM sequence can be found. As stated within the paper, important FDA approved primer amplify regions N1 and N3 of SARS-COV-2 had to be excluded from this technique due to a lack of nearby PAM sequence (Broughton et al., 2020). Thus, it is important to find other conserved regions within the genome that contain PAM sequences if Cas12 protein will be used. Additionally, it is also important to study different Cas12 orthologs that contain different recognizing PAM sequences, as it will allow for a greater scope of target regions (Cebrian-Serrano et al., 2017).

Lateral flow testing strips required for testing of each gene.

One improvement to the efficiency and cost effectiveness of the CRISPER-Cas-12 DETECTOR technique would be to merge testing strips. As of now, highlighted in the paper is the use of a different test strip for each N,E and RNAase P gene in order to increase sensitivity and specificity to come to an accurate diagnosis. Additionally, it requires a control line for each of the test strips, leading to wasted regents. By merging the test strips, the test would have one control line and a testing line for each of the genes. Thereby, allowing for accurate diagnosis with just one strip and lowering the amount of sample and regents required. The use of multiple testing parameters on lateral flow assays are already utilized in antibody testing and should be expanded into this CRISPR test.

Summary of different Cas proteins with their advantages and disadvantages.

While the paper highlights the use of Cas12, there are many other Cas proteins that can be utilized. However, it is beneficial to stick to type II Cas proteins as they require only one large effector protein compared to class I which requires multiple small subunits (Pyne et al. 2016). Cas12 is utilized in the paper by Broughton et al. and sees many benefits compared to other Cas proteins such as Cas13 and Cas14. Cas12 and Cas14 are similar in that they both cut single stranded and double stranded DNA, while also requiring PAM sequences for detection and endonuclease activity, however, no PAM sequence is required for ssDNA in Cas14 (MammothBioscience, 2019). Additionally, they are both fairly accurate and can distinguish between very similar sequences, thus, increasing the sensitivity and specificity of the diagnostic test if utilized (MammothBioscience, 2019). It should be noted that Cas12 is more accurate for double stranded DNA while Cas14 is more accurate for single stranded DNA. Other differences are mainly in the structure of the protein and its required guide RNA (MammothBioscience, 2019). Cas14 is a lot smaller compared to Cas12 and requires less resources when producing, however, Cas14 guide RNA is much larger and requires more resources to produce (MammothBioscience, 2019). Compared to Cas13, which can only cut RNA, there is quite a large difference. Cas13 is unique due to cutting RNA that it does not require a PAM sequence and thus has less stringent requirements for cutting (MammothBioscience, 2019). Furthermore, Cas13 is not very effective in disguishing closely related sequences (MammothBioscience, 2019). Overall, Cas13 is a less accurate cas protein to use. While each of these Cas proteins have their advantages and disadvantages, it is important to know that they are not restricted to one type of diagnostic tests. Even though Cas12 and Cas14 cannot cut RNA, other enzymes can be added to convert RNA to double stranded or single stranded DNA in order to utilize a specific Cas protein (MammothBioscience, 2019). As seen in the paper, where SARS-COV-2 is a positive single stranded RNA virus, but Cas12 is being used due to its great accuracy.

Different CRISPR assay protocols using different equipment, reagents and readout methods.

This technology can be utilized in many other domains of diagnostics and is not limited to COVID-19. As many other CRISPR based detection systems have been created before for viruses other than SARS-COV-2, such as Zika, influence A and HPV (Pardee et al., 2016). CRISPR based detection systems are easily adaptable between different zoonotic viruses, as stated by the paper that creating a SARS-COV-2 based CRISPR system took only 2 week, including validation (Broughton et al., 2020). This technology can therefore be expanded for other upcoming zoonotic viruses which have been the center of pandemics for the last few decades. Increasing diagnostic power for early detection when other technologies such as RT-PCR and ELISA are not yet available or accurate. Furthermore, the technology is starting to be used for non-infectious diseases as well, for example: CRISPR detection systems for certain cancers (Huang et al. 2018) (Vangah et al. 2020). Thereby, projecting the field of diagnostics forward, as we develop more accurate and efficient tests. It should also be noted that CRISPR is at the center of these diagnostic protocols, but can fit into many different assays and thus can be adapted not only by the protein being used but also by the types of regents and readouts. With so much versatility, CRISPR is shaping up to be an easy, cost effective and accurate testing method that can reach many domains of healthcare.

While this technology is advantageous in the realm of diagnostics, it still comes with some limitations. While Point of Care testing is advantageous, the assay requires reagents that are currently limited, leading to less roll out capabilities (University of Oxford Medical Sciences Division, n.d.). The added benefit of having a quick detection time and administration at Point of Care leads to a slightly less sensitive test when compared to RT-PCR (University of Oxford Medical Sciences Division, n.d.). The lowered sensitivity stems from the lack of extensive amplification that is performed during RT-PCR. While the authors did utilize multiplexing by using different guide RNA, they could have increased the sensitivity by multiplexing different cas proteins in order to get better detections (Serrano, et al. 2020). An example of improved assay quality by multiplexing is showcased by comparison of the SHERLOCK v1 and v2 assays (Srivastava et al., 2020). Additionally, the authors could have utilized different Cas proteins or orthologs of Cas12 (differing of PAM recognition sequences) in order to circumvent the issue of lacking PAM sequences for genes N1 and N3 which are outlined in the CDC guidelines (MammothBioscience, 2019) (Cebrian-Serrano et al., 2017). Furthermore, more research can be conducted in other conserved regions of the SARS-COV-2 genome in order to find PAM sequences . Lastly, the assay has a higher limit of detection compared to the CDC assay. This means that it requires more viral copies to be in a sample so that an accurate positive result can be achieved, which increases the likelihood of a false negative.

presentation_two.pptx

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