- Various CRISPR-Based Methods for Testing or Therapeutics Have Been Developed
- The First of These, Termed SHERLOCK, Has Received FDA Approval
- A Current Synopsis of Methods Using CRISPR
The catalyst for this blog was recent media coverage on FDA approval of Sherlock Biosciences’ CRISPR-based diagnostic kit for detection of the SARS-CoV-2 virus, which the company announced on May 7, 2020. This first-ever CRISPR application tests for the presence of the COVID-19 virus.
Sherlock Biosciences cofounders include uber-famous CRISPR-pioneer Feng Zhang, who has been featured in several Zone blogs on various aspects of CRISPR technology. CRISPR continues to draw an incredible amount of attention for gene editing, diagnostics, and basic research, as evidenced by this chart of annual CRISPR-related publications indexed in the NIH PubMed database.
While the total number of these CRISPR publications exceeds 19,000 to date since 2002, mostly over the past 5 years, SARS-CoV-2/COVID-19 is the subject of more than 14,300 publications in only 5 months (January-May, 2020), which reflects a truly astounding rate of response by the global science and biomedical communities in combating COVID-19.
The nexus between CRISPR technology and the current coronavirus pandemic, brought into focus by the aforementioned FDA-approved test, led the Zone to research the literature for this particular test, as well as all other currently available CRISPR-based publications related to SARS-CoV-2 and COVID-19. This blog provides key details of this particular diagnostic method—termed SHERLOCK—and then gives a synopsis of other CRISPR – SARS-CoV-2/COVID-19 publications found in PubMed or posted on the bioRxiv.org website, which has become a trending source of shared “sneak previews” of manuscripts prior to the conventional reviewing process.
In October 2019, Feng Zhang and collaborators published a paper in Nature Protocols titled SHERLOCK: nucleic acid detection with CRISPR, which has received 16,000 views to date and is in the 98th percentile of the 264,738 tracked articles of a similar age in all journals, according to current metrics. This widely read paper provided the step-by-step protocol for a CRISPR-based diagnostic platform that combines existing nucleic acid pre-amplification methodology with novel CRISPR–Cas enzymology for specific recognition of desired DNA or RNA sequences.
This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) in an initial 2017 Science publication by Zhang and collaborators, allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Shortly after its publication, this early report was featured in a Zone blog outlining the initial development of SHERLOCK.
For the purposes of this blog, the following aspects of SHERLOCK are noted and depicted in the simplified scheme shown here. A multicomponent CRISPR RNA (crRNA; black)-Cas enzyme (gray) “RNA targeting complex” is bioinformatically designed to specifically hybridize to the RNA target (blue) of interest, not other RNA background (orange), following an initial pre-amplification step (not shown). This hybridization triggers “collateral RNase activity” directed at a dual labeled fluorophore (F)/quencher (Q) RNA reporter (aka “sensor,” green) that is intramolecularly quenched when intact and emits fluorescence (yellow) when cleaved. Importantly, this collateral RNase activity is reported to be capable of at least 104 turnovers per target RNA recognized, thus lowering the limit of detection.
Alternatively, visual detection in a lateral-flow format can be achieved using a sensor construct with terminal antigen and biotin moieties, as in the COVID-19 virus SERLOCK test discussed in the next section. SHERLOCK is therefore a nucleic acid-based, enzyme-mediated fluorogenic assay, of which there are a variety of existing alternatives, such as TaqMan qRT-PCR. However, SHERLOCK offers a number of advantages, according to the 2019 paper by Zhang and collaborators. These advantages—and some limitations—include the following:
Advantages: SHERLOCK is highly sensitive and specific. It is capable of single-molecule detection in 1-μL sample volumes (2 aM) of both RNA and DNA targets, and single-nucleotide mismatch distinction. The Cas enzyme used in SHERLOCK does not require strict sequence preferences at the target site, allowing more flexibility and a broader target range for SHERLOCK compared to other CRISPR-based variants. An attractive feature of SHERLOCK is the rapid nature of the assay, and the possibility of a single “one-pot” reaction format. Cost per sample is reportedly not problematic.
Limitations: SHERLOCK currently involves the preparation and testing of reaction components, some of which require expertise in protein purification and RNA biology. Moreover, convenient pre-designed assays, including reaction mixtures and RNA/DNA oligonucleotides, are currently not commercially available for SHERLOCK. Also, the multi-step nucleic acid amplification process (see below) may affect precise target quantification, and absolute digital quantification like that in digital droplet PCR is currently not possible.
Protocol for SHERLOCK Detection of COVID-19 Virus
On May 6, 2020, the Broad Institute announced that, to aid the global effort combating the COVID-19 pandemic, Broad Institute of MIT and Harvard, the McGovern Institute for Brain Research at MIT, and partner institutions “have committed to freely providing information that may be helpful, including by sharing information that may be able to support the development of potential diagnostics.”
The announcement added that, “as part of this effort, Feng Zhang, Omar Abudayyeh, and Jonathan Gootenberg have developed a research protocol, applicable to purified RNA, that may inform the development of CRISPR-based diagnostics for SARS-CoV-2, the causative agent of COVID-19.” The face page of this protocol, which can be accessed as a PDF via a link in the announcement, notes that “[t]his protocol should not be used for clinical purposes” due to the fact that developers did not have access to patient samples, and have therefore not been able to validate this assay using them.
For the COVID-19 virus, positive control sequences for the S and Orf1ab gene fragments are specified in the protocol, and can be made by T7 transcription of synthetic oligonucleotide templates.
Using synthetic COVID-19 virus RNA fragments, COVID-19 virus target sequences were consistently detected in a concentration range between 20 and 200 ~10-18 molar (attomolar, aM), which corresponds to 10-100 copies per microliter of input. The test starts with RNA purified from patient samples, as is used for qRT-PCR assays, and can be read out using a dipstick in less than an hour, without requiring elaborate instrumentation. The Zhang lab has posted a 5-min RNA preparation method for detection of COVID-19 virus in a nasopharyngeal swab that is accessible as a PDF at this link.
After RNA extraction from patient samples, the SHERLOCK COVID-19 virus detection protocol works in three steps:
Step (1) – 25 min incubation – isothermal amplification of the extracted nucleic acid sample using a commercially available recombinase polymerase amplification (RPA) kit (see Footnote);
Step (2) – 30 min incubation – detection of pre-amplified viral RNA sequence using Cas13;
Step (3) – 2 min incubation – visual readout of the detection result by eye using a commercially-available paper dipstick.
To check for the presence of COVID-19 viral RNA in nucleic acid extracts of specimens, two targets were chosen from the S gene and Orf1ab gene in the COVID-19 viral genome, depicted here. RPA amplification primers (a forward and reverse pair for each gene) and LwaCas13a CRISPR guide RNAs (crRNAs) were designed for specific detection. In order to maximize the specificity of the assay, guide sequences were selected to minimize off-target hybridization with related human respiratory virus genomes.
Simple, instrument-free and portable detection uses lateral-flow readout by eye via the following RNA reporter comprised of 5’-fluorescein (FAM), 3’-biotin (Bio), 2’-O-methyl A (mA), and ribo (r) A, U, C and G:
5’ FAM-mArArUrGrGrCmAmArArUrGrGrCmA-Bio 3’
On the lateral-flow strips depicted below (see the 2019 report by Zhang and collaborators), a line of streptavidin (SA) will bind to biotin, capturing all the intact probe. Anti-FAM antibodies (Ab) labeled with gold nanoparticles (AuNPs) will bind FAM at the end of the reporter and form a purple color at this first line. When RNA reporters are cleaved by collateral RNase activity because of the presence of target, AuNPs will flow over the SA line to a second line of anti-FAM Ab, thus forming a purple color at the second line that indicates the presence of target. Visualization of these two purple lines indicates a positive test, whereas presence of only the first purple line indicates a negative result.
The published protocol for SHERLOCK recommends performing positive and negative controls alongside tested samples. A negative control could include a water-only input sample for RPA or a sample in which the target molecule is known to be absent. Positive controls include synthetic RNA or DNA targets or purified DNA/RNA in which the target is known to be present. For the COVID-19 virus, positive control sequences for the S and Orf1ab gene fragments are specified elsewhere, and can be made by T7 transcription of synthetic oligonucleotide templates.
Current CRISPR – SARS-CoV-2/COVID-19 Articles in PubMed
As of May 11, 2020, a PubMed search of [(COVID-19) OR (SARS-CoV-2)] AND CRISPR in the Title or Abstract fields gave 12 articles, approximately half of which were review articles commenting on the potential utility of CRISPR technology in the context of CRISPR-based detection or treatment of SARS-CoV-2 or COVID-19, respectively. More importantly, the following articles reported significant experimental results, briefly summarized here.
Broughton et al. developed a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral-flow assay for visual detection of SARS-CoV-2 from nasopharyngeal swab RNA extracts. The method was validated using contrived reference samples and clinical samples from patients in the U.S., including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. This CRISPR-based assay, termed DETECTR, had 95% positive predictive agreement and 100% negative predictive agreement.
The need for scalable technologies to test many samples while simultaneously testing for many pathogens was addressed by Ackerman et al. in the development of Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. Nanoliter droplets containing CRISPR-based nucleic acid detection reagents self-organize in a microwell array to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of >4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, these investigators developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with ≥10 published genome sequences and rapidly incorporates an additional crRNA to detect SARS-CoV-2. In addition, CARMEN’s intrinsic multiplexing and throughput capabilities make it practical to scale, as miniaturization decreases reagent cost per test >300-fold.
Abbott et al. demonstrated a CRISPR-Cas13-based strategy, termed PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. They designed and screened crRNAs targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. A bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the future development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.
Lastly, Davies and Barrangou provided an up-to-date account of the responses to COVID-19 made by seven different research groups representing members of the “CRISPR Community.” This update concluded with the following statement:
“On the research front, it is gratifying to see scientists from so many disparate fields turning their attention to the pandemic, hatching new ideas and building platforms to diagnose, model, screen, and/or treat the disease. An army of scientists is working on solutions, encompassing diagnostics, antivirals, and next-generation vaccines, and willing to make themselves and their resources available as needed.”
Current CRISPR – SARS-CoV-2/COVID-19 Articles in bioRxiv
As of May 11, 2020, a bioRxiv search of [(COVID-19) AND (SARS-CoV-2) AND CRISPR] in the Title or Abstract field gave the following 3 articles, which have self-explanatory titles that can be clicked on to access the unreviewed manuscripts to date.
- An ultrasensitive, rapid, and portable coronavirus SARS-CoV-2 sequence detection method based on CRISPR-Cas12
- SARS-CoV-2 detection with CRISPR diagnostics
- All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus
According to a comprehensive review of the development of RPA by Li et al., the fundamental reaction mechanism of RPA relies on a synthetically engineered adaptation of a natural cellular process called homologous recombination, a key process in DNA metabolism. As depicted below, the standard RPA reaction reagents comprise three key proteins (recombinase, recombinase loading factor, and single-stranded binding protein), which subsequently coordinate with ancillary components such as DNA polymerase, crowding agent, energy/fuel components (e. g. adenosine triphosphate, ATP) and salt to perform the RPA reaction mechanism.
RPA was initially demonstrated as a nucleic acid amplification method for DNA, but it was later shown that RNA could also be a template, by addition of reverse transcriptase (e.g. Murine Leukemia virus (MuLV) reverse transcriptase) and NTPs in the same reaction tube to convert DNA into RNA. A company called TwistDx Inc. sells the commercialized RPA reagents.
As usual, your comments are welcomed.