key: cord-0287224-m0odw194
authors: Rybnicky, Grant A.; Dixon, Radeen A.; Kuhn, Robert M.; Karim, Ashty S.; Jewett, Michael C.
title: Development of a Freeze-Dried CRISPR-Cas12 Sensor for Detecting Wolbachia in the Secondary Science Classroom
date: 2021-10-09
journal: bioRxiv
DOI: 10.1101/2021.10.06.463384
sha: 3869945e4fb6b622ae430bfc5d53df3594c22778
doc_id: 287224
cord_uid: m0odw194

Training the future synthetic biology workforce requires opportunity and exposure to biotechnology concepts and activities in secondary education. Detecting Wolbachia bacteria in arthropods using PCR has become a common way for secondary students to investigate and apply DNA technology in the science classroom. Despite this framework, cutting-edge biotechnologies like CRISPR-based diagnostics have yet to be widely implemented in the classroom. To address this gap, we present a freeze-dried CRISPR-Cas12 sensing reaction to complement traditional DNA technology education and teach synthetic biology concepts. The reactions accurately detect Wolbachia from arthropod-derived PCR samples in under 2 hours and can be stored at room temperature for over a month without appreciable degradation. The reactions are easy-to-use and cost less than $40 to implement for a classroom of 22 students including the cost of reusable equipment. We see this technology as an accessible way to incorporate synthetic biology education into existing biology curriculum, which will expand biology educational opportunities in science, technology, engineering, and mathematics (STEM) education.

We set out to develop a CRISPR-Cas12 diagnostic for Wolbachia detection to implement in secondary classrooms. First, we developed a functional CRISPR-Cas12 diagnostic. Cas12, like other CRISPR nucleases, binds and cleaves DNA at a specific sequence encoded by a CRISPR RNA (crRNA) 17 . Once activated, Cas12 indiscriminately cleaves single stranded DNA (ssDNA) in solution ( Figure 1B) 15 . This indiscriminate cleavage activity can be visualized within a diagnostic in a variety of ways, including the use of a ssDNA chemically modified with a fluorophore on one end and a fluorescence quencher on the other. In the uncleaved state, the ssDNA probe emits no fluorescence upon excitation as the quencher molecule is near the fluorophore and absorbs the fluorescent emission from the fluorophore. Once cleaved, the ssDNA probe is no longer intact, and the fluorescence quencher is not close enough to the fluorophore to absorb the fluorescent emission. To test for CRISPR-Cas12 activity, we screened all possible crRNAs that can target the consensus 483 bp Wolbachia amplicon previously developed for the Wolbachia Project. Of the five locations with the appropriate TTTV Protospacer Adjacent Motif (PAM) 17 (Supplementary Figure S1 ), all possible crRNAs mediated detection of the Wolbachia amplicon ( Figure 1C ). Although all were functional, reaction endpoint fluorescence varied with different crRNA constructs. crRNA 286R yielded the highest endpoint fluorescence as well as fold activation. Thus, we selected crRNA 286R for further development.

With a functional CRISPR-Cas12 diagnostic for Wolbachia detection in hand, we next wanted to assess the possibility of freeze-drying the system. Previous works have shown that cell-free systems, including CRISPR-based diagnostics, can be freeze-dried for increasing stability and portability 18, 19 . Such features would be advantageous for preparation and delivery in the classroom setting. We freeze-dried CRISPR-Cas12 cell-free reactions in a VirTis BenchTop Pro lyophilizer at ≤100 mtorr and −80 °C overnight or until fully freeze-dried. We then compared CRISPR-Cas12 reactions prepared from fresh reagents and these pre-assembled, lyophilized reactions to determine whether the CRISPR-Cas12 formulation is stable through the lyophilization process. Unfortunately, when lyophilized, the CRISPR-Cas12 formulation demonstrated no activity in the presence of activating DNA (Figure 2A ). We hypothesized that the lack of activity was due to degradation of the Cas12 protein or the crRNA.

To address this bottleneck and improve the stability of the reactions, we evaluated diagnostic activity when reactions were lyophilized in the presence of different lyoprotectant formulations ( Figure 2B ). Specifically, we tested sucrose, trehalose, and dextran (70 kDa average weight) individually and in combination. We found that the activity of reactions supplemented with sucrose and trehalose individually increased with concentration, but dextran exhibited a concentration optimum of 25 mg/mL in the final reaction. Although combining 25 mg/mL each of sucrose and trehalose achieved similar endpoint fluorescence as 50 mg/mL of each respective lyoprotectant, combining 25 mg/mL of either sugar with 25 mg/mL dextran increased the retained activity. Combining 25 mg/mL each of sucrose and dextran exhibited approximately 75% recovery of the pre-lyophilization signal.

Additionally, all formulations containing dextran exhibited beneficial changes in freezedried pellet consistency. Rather than a powdery pellet subject to movement by static electricity, all lyophilized dextran formulations exhibited a bulky, solid pellet unaffected by static charges. This made manipulation of pellets much easier and decreased the risk of static electricity ejecting portions of the pellet from the tube. In making the pellet easier to work with, the inclusion of dextran in the lyoprotectant formulation made the FD-CC12 reactions more amenable to use in secondary science classrooms. Moving forward we chose to use 25 mg/mL each of sucrose and dextran to formulate our freeze-dried reactions.

We next characterized the final formulation on freeze-dried CRISPR-Cas12 (FD-CC12) reaction stability over time. After lyophilization we stored reactions at either 20 or 37 °C in vacuum sealed bags with a desiccant card for two months, monitoring activity over time. When stored at room temperature, the lyophilized reactions retained approximately 75% activity in the on state after 2 months of storage without appreciable leak in off state ( Figure 2C ). When stored at 37 °C for the same amount of time, the reactions retained over 40% activity in the on state without leak.

In the Wolbachia Project, students amplify DNA using PCR following DNA extraction from arthropods. Different classrooms may use different PCR reaction mixtures, based on price and preference. To assess generalizability of our FD-CC12 reactions and facilitate integration into existing classroom activities, we therefore tested compatibility with common PCR reaction mixtures. We evaluated whether several commonly used PCR kits (Q5 Hotstart, NEB Taq, MiniPCR EZ PCR, Thermo DreamTaq, and Promega GoTaq) could be used to rehydrate the FD-CC12 reactions and maintain functionality. Final endpoint fluorescence varied among FD-CC12 reactions rehydrated with different PCR kits, all achieving at least 60% of the signal achieved by reactions rehydrated with purified DNA ( Figure 2D ). Despite this variability in endpoint fluorescence, all the reactions produced observable signal in the on state and negligible signal in the off state. These data highlight the robustness of our FD-CC12 diagnostic for detecting Wolbachia.

With stable and robust FD-CC12 reactions established, we next wanted to test compatibility with the Wolbachia Project educational framework. We first wanted to make sure that Wolbachia DNA from arthropod-derived samples could be detected. When rehydrated with a PCR reaction that used bulk DNA from a Wolbachia infected Nasonia vitripennis wasp as template, the FD-CC12 reaction achieved endpoint yields comparable to those observed in the PCR kit screen ( Figure 3A ). Using a Wolbachia-free N. vitripennis wasp-derived PCR, the FD-CC12 reaction showed signal above background observable by fluorescent plate reader but not by eye.

Once validated with control insects, we next assessed the ability of the workflow to be completed in a high school classroom setting by a high school student. FD-CC12 reactions were prepared at Northwestern University and shipped to Centennial High School (CHS) in Roswell, Georgia. At CHS, a student collected and identified 22 arthropods across 6 orders (Supplementary Table S2 ), extracted bulk DNA from each specimen, and performed PCR to amplify the Wolbachia amplicon as is standard practice in the Wolbachia Project workflow. Those PCR products were then used to rehydrate FD-CC12 reactions and were also visualized using gel electrophoresis. Out of the 22 samples tested, 21 agreed between FD-CC12 reaction and gel electrophoresis ( Figure 3B ). For sample ID# 11, the gel indicated Wolbachia infection while the FD-CC12 reaction indicated no infection. Upon sequencing the Wolbachia amplicon for sample ID# 11, it was revealed that there were 5 mutations within the protospacer region the crRNA 286R targeted (Supplementary Figure S2) . As Wolbachia are a diverse genus of bacteria, it is not unreasonable to expect that some isolates would evade detection by any single crRNA. Using multiple crRNAs in the same reaction to create OR gate logic, it may be possible to detect a wider diversity of isolates. Given the compatibility of the FD-CC12 reactions with arthropod-derived samples, successful completion by a high school student, and high accuracy of sensing reactions, this demonstrates that FD-CC12 reactions can be integrated into the Wolbachia Project laboratory activities.

We developed a FD-CC12 Wolbachia DNA sensing reaction that can be used to teach synthetic biology concepts in the secondary science classroom. As designed, the sensing reactions detect a PCR amplicon commonly used to diagnose Wolbachia infection in fieldcollected arthropods as a demonstration of DNA technologies ( Figure 1A) . We identified and evaluated 5 functional crRNAs that can be used to detect this amplicon with Cas12 ( Figure 1C ). Additionally, we identified a lyoprotectant additive formulation that both recovers about 75% of reaction endpoint fluorescence after lyophilization ( Figure 2A&B ) and is stable at room temperature for over a month without appreciable degradation ( Figure 2C ). Using this formulation, we evaluated the reaction's compatibility with commonly used PCR kits, finding that the freezedried reactions could be rehydrated with virtually any unpurified PCR product and still distinguish the presence of the Wolbachia amplicon in the sample ( Figure 2D ). To validate the reaction can be applied to the established classroom activity, we rehydrated freeze-dried reactions with arthropod-derived samples. Nasonia wasps with known Wolbachia infection states yielded results as expected ( Figure 3A) . Screening of 22 field-collected arthropods across 6 orders for Wolbachia infection by a high school student using gel electrophoresis and FD-CC12 reactions yielded a 95% accuracy rate ( Figure 3B ).

While we recommend that FD-CC12 sensing reactions be used to complement gel electrophoresis in DNA technology education, we do recognize that specific features of our sensing reactions may make them a more attractive readout than gel electrophoresis to secondary science educators. The FD-CC12 reactions primarily lower two barriers DNA visualization in the classroom: 1) FD-CC12 reactions are less technically difficult to set up and run than gel electrophoresis and 2) FD-CC12 reactions are less expensive. While commonplace in the molecular biology research lab, gel electrophoresis poses a few technical hurdles to implementation that science educators may face. Foremost, gel electrophoresis is intimidating to educators who have not had hands-on training in a laboratory or elsewhere. The ability to properly prepare and cast an agarose gel, pipette samples into the well of a gel, and consistently stain and visualize gels takes hands-on practice before it becomes routine. Collectively, these technical hurdles may cause teachers to seek more expensive alternatives (precast gels for example) or to avoid implementation of gel electrophoresis altogether. Alternatively, the FD-CC12 reactions contain all necessary components within a single tube, are easier to pipette analyte into, and are visualized by handheld blue light imager without the addition of stains. The use of freeze-dried cell-free reactions has the potential to increase teacher confidence in their laboratory skills and encourage them to attempt laboratory activities they may have been hesitant to try before like gel electrophoresis. Similarly, the initial monetary investment required to implement FD-CC12 sensing in the classroom is lower than implementing gel electrophoresis. Requiring only incubation at 37 °C and a blue light imager like the MiniPCR P51™ Molecular Fluorescence Viewer, the FD-CC12 reactions have a startup cost of ~$38 for a 22-student class. Alternatively, the equipment to cast gels, generate current, house the gel in buffer, and visualize the gel as well as initial reagents cost ~$1000 or ~$300 for the same sized class for traditional or education optimized gel electrophoresis equipment, respectively (Table 1 ). Assuming one sample per student and class size of 22 students, the CRISPR-Cas12-based activity is cheaper to run for 848 students or fewer than education optimized gel electrophoresis set up. The price of FD-CC12 reactions can be further reduced about 10X by applying state-of-the-art cell-free technology like the use of paper instead of tubes 20 . That being said, gel electrophoresis systems are generalizable to visualizing almost any DNA sample while the FD-CC12 sensing reaction presented is specific to Wolbachia amplicon detection given the crRNA used.

Although we focused on detection of Wolbachia in the secondary science classroom, we note that the technology described has potential applications beyond the scope presented. Within science education, the DNA that is detected can easily be changed by altering the crRNA in the reaction. There are numerous well established laboratory activities that use gel electrophoresis to detect double stranded DNA PCR product. Given the presence of a functional PAM sequence in the DNA analyte and appropriate design of a crRNA, the technology described here can be extended to other educational activities beyond Wolbachia detection. The technology is not limited to the classroom either. As the freeze-dried cell-free reactions are still functional after 56 days storage at 37 °C ( Figure 2C) , it is feasible to use these reactions in on-site biological field research. In addition, FD-CC12 reactions could be implemented in field stations where gel electrophoresis may be difficult or impractical. As mentioned before, other DNAs can alternatively be detected by exchanging the crRNA in the system. This can lead to applications such as tracking of specific organisms in the environment by DNA detection, identification of visibly indistinguishable species, or monitoring the spread of mobile genetic elements in the environment.

Taken together, FD-CC12 reactions have the potential to be a simple, cost-effective companion to gel electrophoresis that provide a hands-on way to teach CRISPR-based nucleic acid detection and concepts of synthetic biology. In integrating this activity with the established DNA technology workflow designed by the Wolbachia Project, we anticipate widespread adoption of CRISPR-Cas12-based detection into the classroom, further strengthening the effort to expose school age children to synthetic biology concepts and priming the future STEM workforce.

Fresh Cas12 reactions were assembled by mixing reaction buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3), 45 nM AsCas12a V3 (IDT), 45 nM crRNA (IDT), 1 µM ssDNA Fluorophore-Quencher (FQ) Probe (IDT), and lyoprotectant. Prior to addition of FQ Probe and lyoprotectant, the mixture was equilibrated at 37 °C for 15 minutes. Upon addition of the analyte, the reaction was incubated at 37 °C and fluorescence was measured (490 nm Excitation, 525 nm Emission) using a Synergy H1 microplate reader (BioTek, USA) and Gen5 v. 2.09 (BioTek) software.

Lyophilized Cas12 reactions were assembled as described above and then flash frozen with liquid nitrogen in PCR strip tubes (Thermo Scientific, AB2000) with a small hole melted in the cap of each tube. The reactions were lyophilized using a VirTis BenchTop Pro lyophilizer (SP Scientific) at ≤100 mtorr and −80 °C overnight or until fully freeze-dried. Following lyophilization, tubes were packaged in Food Saver® bags with a desiccant card, vacuum sealed and stored at the indicated temperature. When ready for use, tubes were removed from the Food Saver® bags and rehydrated with the same volume of analyte as the mixture prior to lyophilization. Reactions were incubated at 37 °C and fluorescence was measured as described above.

Arthropods belonging to six different orders (Supplemental Table S2 ) were collected from Roswell, GA and Brevard, NC. Each specimen was preserved in 75% EtOH in a 1.5mL collection tube and frozen at -20 °C. Samples were visually identified to taxonomic order and later identified further by COI barcoding. PCR using the primers described by Folmer 21 was performed and the product was sanger sequenced (GeneWiz). Consensus sequences for each specimen were obtained using DNA Subway (CyVerse/DNA learning Center) and NIH nucleotide BLAST 22 was used to taxonomically identify the arthropod. Wolbachia-infected and Wolbachia-free Nasonia vitripennis wasps were obtained from the Wolbachia Project through the Bordenstein Lab at Vanderbilt University. Bulk DNA was extracted from each specimen by manual grinding using sterile, plastic minipestles for 2 minutes in a 1.5 mL microcentrifuge tube containing 200 µL of lysis buffer/EDTA mix and subsequent purification with the Monarch Genomic DNA Purification Kit (catalog #T3010) as directed. A 100µL DNA elution was collected stored at -20 °C until used for polymerase chain reaction (PCR).

PCR was performed using 2µL of eluted DNA, Wspec-F (5'−CAT ACC TAT TCG AAG GGA TAG−3') and Wspec-R (5'−AGC TTC GAG TGA AAC CAA TTC−3') primers and MiniPCR EZ PCR Master Mix (catalog RG-1000-01). MiniPCR Mini8 thermocyclers were used for PCR with the following protocol: initial denaturation at 94 °C for 120 sec and 34 cycles of denaturation 95 °C for 30 sec, annealing 55 °C for 45 sec, extension 72 °C for 60 sec and final extension 72 °C for 600 sec. PCR product was frozen at -20 °C for further use.

Gel electrophoresis assays were completed in a secondary science education classroom at Centennial High School in Roswell, GA using the MiniPCR blueGel™ electrophoresis system with built-in transilluminator. Gels were poured using GelGreen® Agarose Tabs™ to a concentration of 2% agarose. 5 µL of each PCR product was loaded into a well and were electrophoresed for 30 min. The presence of Wolbachia in the arthropod specimen was determined by presence of a 438 bp amplicon using the built in blue light transilluminator.

Freeze-Dried Cas12 reactions were assembled and packaged at Northwestern University in Evanston, IL. The reactions were then shipped at room temperature to Centennial High School in Roswell, GA. Reactions were stored at room temperature for 9 days prior to rehydration. PCR strip tubes containing a freeze-dried pellet of the CRISPR-Cas12 system were rehydrated using 10 µL of PCR product. Tubes were spun briefly at 10,000 rpm and moved to a heat block to

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We would like to acknowledge MiniPCR for their contribution of materials and reagents. We would also like to thank Sarah Bordenstein and the Wolbachia Project for helpful conversation and for N. vitripennis specimens. We would also like to acknowledge the Centennial High School DNA Club for providing collaborative biology research opportunities for high school students. 

 Figure 1 . CRISPR-Cas12 diagnostics can be integrated into existing high school biology curriculum. A) Cas12-based DNA sensing reactions can theoretically be used in addition to gel electrophoresis to detect Wolbachia infection in student collected arthropod samples. B) Cas12 is activated by the presence of DNA complementary to the crRNA and indiscriminately cleaves ssDNA. A ssDNA quencher-fluorophore probe can convert this activity to observable fluorescence. C) All possible crRNAs against the Wolbachia amplicon were tested for activity. Endpoint fluorescence after incubation at 37 °C for 2 hours is reported. Error bars represent standard deviation of 4 replicates. Purified synthetic DNA with the same sequence as the consensus Wolbachia amplicon was used as an activator.