, Fire blight, caused by the gram-negative bacterium Erwinia amylovora, is one of the most devastating diseases affecting members of the Rosaceae family, including apples and pears (Malnoy et al., 2012; Vanneste et al., 2000). The pathogen is highly invasive and rapidly systemic, causing water-soaked lesions, ooze production, necrosis, and the characteristic “burned” appearance of shoots and branches (Vanneste et al., 2000). Its rapid systemic spread is driven by a type III secretion system that injects effector proteins such as harpins into host cells to promote infection (Kim et al., 1997; Kim and Beer, 1998). Once established, E. amylovora spreads quickly under warm and humid conditions, making outbreaks difficult to contain (Vanneste et al., 2000). As there are no curative treatments, management relies heavily on predictive modeling, preventive antibiotic sprays, and the immediate removal of infected trees (Park et al., 2017), practices that are costly and environmentally burdensome. Consequently, early and reliable detection remains the most important tool for preventing large-scale losses.
In Korea, the threat of fire blight has intensified since its first official detection in 2015 (Myung et al., 2016; Park et al., 2016). Following the initial outbreak, comprehensive genomic analyses of the E. amylovora strains in Korea have revealed important genetic characteristics that can be utilized in surveillance and diagnostic development (Song et al., 2021; Song et al., 2025). The introduction of E. amylovora into an agricultural landscape where Erwinia pyrifoliae, the causative agent of black shoot blight in Asian (Kim et al., 1999; Rhim et al., 1999), is already endemic complicates disease surveillance. These two species cause nearly indistinguishable symptoms (Kim et al., 2001a; Kim et al., 2001b) and share high genomic similarity (Gardner and Kado, 1972; McGhee et al., 2002), making conventional diagnostic methods, such as culture-based identification, biochemical assays, and even some PCR approaches, prone to ambiguity or limited in specificity. As the scale and recurrence of fire blight outbreaks in Korea have increased, the need for rapid, accurate, and field-adaptable molecular diagnostics capable of differentiating closely related Erwinia species has become urgent.
Nucleic acid-based assays, including conventional PCR, real-time qPCR (Gottsberger, 2010; Jin et al., 2023; Pirc et al., 2009; Taylor et al., 2001), and loop-mediated isothermal amplification (Bühlmann et al., 2013), have greatly improved fire blight diagnostic sensitivity and specificity. However, these methods still require laboratory infrastructure, trained personnel, and thermal cycling equipment. They also commonly rely on DNA extraction, which hampers their utility for point-of-care or on-site testing in orchards. Additionally, because E. amylovora and E. pyrifoliae differ by only a limited number of single nucleotide polymorphisms (SNPs) across their genomes, designing primers or probes that reliably discriminate these species remains a persistent challenge.
CRISPR-based diagnostics have emerged as a powerful alternative due to their exceptional sequence programmability (Mojica et al., 2005) and ability to operate under simple reaction conditions. Systems based on Cas12 (Chen et al., 2018) and Cas13 (Abudayyeh et al., 2016) enzymes leverage collateral cleavage of fluorescent or lateral flow reporter molecules upon recognition of a target sequence. Among these, Cas13a is particularly attractive because it directly senses RNA molecules without requiring DNA amplification. Direct detection of mRNA eliminates the need for both DNA amplification and in vitro transcription, thereby simplifying the workflow and reducing reaction times. Also, while many DNA targets are present as single-copy genes, mRNA molecules typically exist in multiple copies within a cell, increasing the likelihood of detection.
Despite these advantages, CRISPR-Cas13a diagnostics have typically been applied in viral plant pathogens (Marqués et al., 2022; Zhang et al., 2021) and rarely used to detect bacterial phytopathogens (Khmeleva et al., 2024), and to date, no studies have explored its use for detecting E. amylovora or distinguishing it from E. pyrifoliae. Additionally, previously developed CRISPR-Cas13a-based plant pathogen detection systems includes a nucleic acid extraction step that is both time-consuming and requires expertise. The feasibility of extraction-free CRISPR detection, an approach that aims to minimize sample processing and accelerate detection, remains largely unexplored for bacterial plant pathogens.
To address these gaps, this study identifies E. amylovora-specific (EA-specific) genetic biomarkers, designs and evaluates CRISPR RNAs (crRNAs) targeting discriminatory SNPs, and develops a Cas13a-based detection workflow optimized for rapid, specific, and extraction-free detection from plant material. Together, these efforts aim to expand CRISPR-based tools in plant disease diagnostics and provide foundational strategies for field-ready detection of bacterial pathogens with high genomic similarity. This extraction-free detection system has the potential to expand field diagnostics and substantially strengthen biomanagement strategies for fire blight disease.
Materials and Methods
Bacterial strains and culture conditions
E. amylovora TS3128 and E. pyrifoliae Epk1/15(81) were used as representative strains for each species. TS3128, isolated during the first fire blight outbreak in Anseong, South Korea (Kang et al., 2021), is widely used in Korean fire blight research. Epk1/15(81), collected in 2014 from apple trees exhibiting black shoot blight symptoms (Lee et al., 2018), represents currently known E. pyrifoliae strains targeted by the designed crRNAs. All work involving E. amylovora was conducted in the BSL-2 laboratory under containment conditions.
Escherichia coli BL21 (DE3) strains used for Cas13a expression were cultured in lysogeny broth (LB; 1% tryptone, 0.05% yeast extract, 1% NaCl) supplemented with ampicillin at 37°C. E. amylovora TS3128 and E. pyrifoliae Epk1/15(81) were grown in LB at 30°C. All strains were first streaked onto LB agar and incubated overnight at their respective temperatures. Seed cultures (2 mL) were prepared from single colonies and grown overnight with shaking. For experiments, main cultures (20-25 mL) were inoculated from seed cultures and incubated until reaching mid-log phase (optical density at 600 nm [OD600] = 0.5-0.6).
Cas13a expression and purification
The pC013 Twinstrep-SUMO-huLwCas13a plasmid (Addgene plasmid #90097; http://n2t.net/addgene:90097; RRID: Addgene_90097) (Gootenberg et al., 2017) was transformed into E. coli DH5α for plasmid propagation and E. coli BL21(DE3) for expression. pC013 - Twinstrep-SUMO-huLwCas13a was a gift from Feng Zhang (Addgene plasmid # 90097; http://n2t.net/addgene:90097; RRID: Addgene_90097). Transformants were selected on ampicillin supplemented LB agar plates, plasmids were purified and sequence-verified.
For expression, E. coli BL21(DE3) cultures were initiated from a single colony, grown overnight, and expanded into LB until mid-log phase (OD600 ≈ 0.5). Cas13a expression was induced with 0.1 mM IPTG for 3 h at 37°C. Cells were harvested, resuspended in PBS, and lysed by sonication. Supernatants were obtained by centrifugation.
Cas13a was purified using Ni-NTA agarose resin (Qiagen, Hilden, Germany) pre-equilibrated with 20 mM Tris-NaCl (pH 7.5) buffer. After washing with 5 mM imidazole, proteins were eluted with increasing imidazole concentrations (20, 200, 500 mM). Elution fractions were assessed by SDS-PAGE. Purified protein was mixed 1:1 with 2X storage buffer (0.1 M Tris-HCl [pH 7.5], 1.2 M NaCl, 10% [w/v] glycerol, and DEPC-treated water), quantified by Bradford assay, aliquoted, and stored at −80°C.
crRNA design and synthesis
For crRNA design, we acquired the target gene sequences from the NCBI database. E. amylovora ATCC 49946 and E. amylovora CFBP 1430 were used as representative E. amylovora sequences as they commonly used as reference sequences. In addition, the E. amylovora FB-86 strain, was used as a representative of Korean-E. amylovora strains as the Korean strains are genetically nearly identical amongst themselves (Song et al., 2021). E. pyrifoliae Ep1/96, DSM 12163, EpK1/15, and Ejp617 sequences were used for comparison. Lastly, the E. tasmaniensis Et1/99 sequence was used to find E. amylovora-specific sequences.
Candidate crRNAs were generated using the Cas13adesign platform (https://cas13design.nygenome.org/) (Guo et al., 2021; Wessels et al., 2020), which scores guides based on predicted folding energy, local sequence context, and target accessibility. Guides with standardized scores ≥0.70 were retained. Candidates were grouped by their position along the target RNA (5′, middle, 3′), and for each region, the crRNA carrying the greatest number of EA-specific SNPs, particularly within the seed region (a mismatch-sensitive binding segment of the spacer; bases 5-15) of the crRNA (Gootenberg et al., 2018), was selected. Final crRNA sequences and corresponding in vitro transcription templates are provided in Supplementary Table 1.
Duplex DNA template oligonucleotides for each crRNA were synthesized (Macrogen, Seoul and Bioneer, Daejeon, Korea). crRNAs were transcribed in vitro using the HiScribe® T7 Quick High Yield RNA Synthesis Kit (NEB, Ipswich, MA, USA) in a 30 μL reaction, which was subsequently diluted to 50 μL with DEPC-treated water. Template DNA was removed by two sequential DNase I (NEB) treatments (37°C, total 30 min). The crRNA was purified with the Monarch® RNA Cleanup Kit (NEB). crRNA concentration and integrity were assessed by Nanodrop and 10% denaturing urea-PAGE after heat denaturation at 95°C for 5 min.
Full-length target RNA preparation
PCR primers complementary to the target genes were designed and synthesized to have T7 overhangs needed for in vitro transcription (Supplementary Table 2). Full length target genes were amplified with the Pfu DNA polymerase (Promega, Madison, WI, USA) with traditional PCR conditions. Amplified DNA targets were used as templates for subsequent invitro synthesis similar to the crRNA synthesis with an adjustment of the final volume to 20 μL, as recommended by the manufacturer. Remaining DNA were degraded using the DNase I provided in the kit. RNA purification was performed with the Monarch® RNA Cleanup Kit (NEB) according to the instructions provided by the manufacturer. The purified RNA was subject to a Nanodrop analysis and verified with 10% denaturing urea polyacrylamide gel electrophoresis after a 5-minute denaturation step at 95°C.
CRISPR-Cas13a detection assay
Cas13a reactions (20 μL total) contained 0.02 M HEPES (pH 6.8), 9 mM MgCl2, Cas13a (0.3 μg), RNase inhibitor, crRNA (30 ng), and a fluorescent reporter (10 pmol; [FAM]-5′-UUUUU-3′-[BHQ1]). One microliter of target RNA was added to a 19 μl reaction mix. For each experiment, four mixes were prepared and aliquoted into three technical replicates. Fluorescence was measured on a QuantStudio 3 (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C, with FAM signals corrected using ROX as an internal reference. Incubation times are provided in the corresponding figure legends.
Buffer optimization for extraction-free detection
An initial lysis buffer consisting of 0.02 M HEPES, 1 mM EDTA, 0.1 M DTT, and w/v 0.05% Triton X-100, supplemented with lysozyme was first tested for extraction-free CRISPR-Cas13a detection. Based on performance, a final lysis buffer consisting of 0.5 N NaOH supplemented with 0.1 M DTT and 1X protease inhibitor (GenDEPOT, Katy, TX, USA) was selected for subsequent assays.
For bacterial preparation, E. amylovora cultures were seeded in 2 mL LB broth and incubated overnight at 30°C. Main cultures were inoculated at an initial OD600 of 0.05 in 50 mL LB and grown for 6 h at 30°C before harvesting. Cells were washed twice with 1× PBS and resuspended in distilled water to a final OD600 of 10.
For sample preparation, 100 μL of the cell suspension was aliquoted into homogenizer tubes containing beads (MP Biomedicals, Irvine, CA, USA), either alone or with 0.05 g of Malus domestica leaf disks prepared using a 10 mm leaf borer. Lysis buffer was added to a final volume of 900 μL, and samples were homogenized for 30 seconds, followed by neutralization with 100 μL of 0.5 N HCl. The mixtures were vortexed, centrifuged, and the supernatants were used directly as inputs for the CRISPR-Cas13a detection assay.
Results
CRISPR-Cas13a-based detection targeting the 16S V3 region of E. amylovora
The 16S rRNA gene is generally a highly conserved and abundantly transcribed locus in bacteria, making it an advantageous target for nucleic acid-based diagnostics. Its stable expression and high copy number ensure robust transcript availability and the gene was also among the first targets for the utilization of the CRISPR-Cas13a system for detection (Gootenberg et al., 2017). Based on these features, we selected the 16S rRNA gene as the initial target for developing our amplification- and extraction-free detection system.
The 16S rRNA gene sequences of E. amylovora and E. pyrifoliae are highly similar and have only a small number of SNPs. The V3 region of the 16S rRNA gene is a hypervariable region that is commonly used for taxonomic discrimination, and by leveraging an EA-specific SNP within the V3 region, we designed a crRNA capable of discriminating E. amylovora from E. pyrifoliae (Fig. 1A). Full-length target RNAs were synthesized in vitro to evaluate the performance of the CRISPR-Cas13a detection system. To rule out false-positive signals, we tested reactions containing different combinations of Cas13a protein, the V3-targeting crRNA, and the synthetic full-length RNA. Reactions containing Cas13a exhibited slightly elevated background fluorescence compared to those lacking the protein, suggesting a minimal level of nonspecific collateral activity (Fig. 1B). However, this background remained low, and a distinct fluorescence signal was observed only when all three components, Cas13a, crRNA, and target RNA, were present. These results confirm that Cas13a activation occurred specifically through cognate crRNA-target recognition.
Amplification-free detection of E. amylovora with high activity crRNAs
To identify additional gene targets suitable for Cas13a-based detection, we surveyed housekeeping genes of E. amylovora known to exhibit stable and abundant expression. Based on previous transcriptomic and genomic references, four conserved loci, ffh, gyrA, proC, and recA (Kałużna et al., 2017), were selected for crRNA design. For each gene, we designed three crRNAs positioned near the 5′ end, middle, and 3′ end of the coding sequence to account for potential positional effects on target accessibility (Fig. 2A). Full-length RNA targets corresponding to each gene were synthesized in vitro and used to evaluate crRNA activity across a range of RNA concentrations. Using the Cas13a fluorescence reporter assay, we assessed the efficiency and sensitivity of all designed crRNAs under identical reaction conditions.
The CRISPR-Cas13a system consistently detected as little as 10 fmol of synthetic target RNA within 5 minutes of reaction initiation (Fig. 2B), demonstrating robust sensitivity and rapid signal generation. Performance varied among individual crRNAs, enabling selection of the most active guides for further development. The top-performing crRNAs for each successfully detected gene were ffh287, proC69, and recA547. In contrast, all three gyrA-targeting crRNAs showed markedly lower activity and failed to reach the performance threshold observed for the other targets and was therefore excluded from further downstream validation. Together, this systematic design and screening approach identified multiple high-activity crRNAs across the four E. amylovora housekeeping genes, expanding the pool of reliable targets for the Cas13a diagnostic platform.
Species-level discrimination of E. amylovora using CRISPR-Cas13a
To evaluate species specificity, we tested the crRNAs ffh287, proC69, recA547 along with the V3 crRNA against full-length target RNAs synthesized from E. amylovora and E. pyrifoliae. Among the candidates, the proC69 crRNA failed to discriminate between the two species (Fig. 3A). Sequence inspection revealed that the seed region, a region on the crRNA that is critical for distinguishing mismatches, of the proC69 crRNA differed by only a single nucleotide between E. amylovora and E. pyrifoliae (Fig. 2A), likely insufficient for robust Cas13a-mediated discrimination. Therefore, the proC69 crRNA was excluded from further validation.
While the initial evaluations relied on synthetic RNA, we next tested the ability of each crRNA to detect native transcripts extracted from bacterial cultures (Fig. 3B). Under these biologically relevant conditions, ffh287 also failed to achieve species specificity. The fluorescence signal generated in reactions containing E. amylovora mRNA was substantially lower than expected based on its previous in vitro performance, suggesting that reduced positive signal amplitude, rather than elevated background, accounted for the loss of discriminatory power.
In contrast, both recA547 and the V3 crRNA produced strong, EA-specific fluorescence signals when tested with bacterial mRNA, with minimal activation in the presence of E. pyrifoliae transcripts (Fig. 3B). These results demonstrate that recA547 and the V3 crRNA maintain high specificity in detecting E. amylovora across synthetic and native RNA targets and therefore represent the most reliable guide RNAs for downstream diagnostic development.
Extraction-free detection of E. amylovora in plant samples
To develop an extraction-free diagnostic workflow capable of detecting E. amylovora directly from plant tissues, we prepared Malus domestica, natural hosts of fire blight, leaf disks with cultured bacterial cells. We first evaluated a mild lysis buffer containing HEPES, EDTA, lysozyme, and DTT. This formulation was selected to gently disrupt bacterial cells while preserving RNA integrity. HEPES provides pH stability, EDTA chelates divalent cations and destabilizes the outer membrane (Leive, 1968), lysozyme hydrolyzes the peptidoglycan layer of Gram-negative bacteria (Chipman and Sharon, 1969), and DTT to prevent cysteine oxidation and preserve the catalytic conformation of the Cas13a protein (Cleland, 1964). Despite this rationale, EA-specific detection was unsuccessful (Fig. 4A). Fluorescence signals were indistinguishable across mock, E. pyrifoliae, and E. amylovora samples, regardless of whether plant disks were included. Moreover, fluorescence from the E. amylovora cell lysate condition was no higher than the mock or E. pyrifoliae controls, suggesting that bacterial lysis was insufficient and RNA was not released into the reaction.
Seeking a more robust lysis strategy, we next tested an NaOH buffer (Audy, 1996), a strong base capable of rapidly disrupting bacterial membranes and denaturing cellular components, supplemented with DTT and protease inhibitor. Screening a range of NaOH concentrations revealed that 0.5 N NaOH effectively lysed E. amylovora cells and enabled clear EA-specific fluorescence using extraction-free cell suspensions (Fig. 4B). Although NaOH facilitated RNA release, the Cas13a protein typically works best in a 6.8 pH environment (Knott et al., 2017), therefore, we evaluated whether subsequent neutralization could improve detection sensitivity (Sun et al., 2025). Neutralizing the lysate with an equivalent concentration of HCl substantially improved the positive signal relative to NaOH treatment alone (Fig. 4C). Based on these results, we finalized the lysis procedure as sequential treatment with 0.5 N NaOH followed by neutralization with 0.5 N HCl.
Using this optimized lysis buffer, we tested extraction-free detection of E. amylovora in the presence of plant material. The system successfully detected E. amylovora within 10 minutes using the V3 crRNA, demonstrating robust, plant-compatible performance without the need for nucleic acid purification or amplification (Fig. 4D). Additionally, since the buffer screening had initially been performed with the V3 crRNA alone, we further evaluated the remaining candidate crRNAs under the optimized conditions. Although ffh287 showed slight separation between E. amylovora and E. pyrifoliae, the difference was not statistically significant (Fig. 4E). In contrast, recA547 produced clearer species-specific activation and stronger fluorescence, consistent with its performance in earlier assays (Fig. 4F). These results establish a rapid, extraction-free lysis protocol capable of supporting Cas13a-based detection directly from plant samples and identify V3 and recA547 as the most reliable crRNAs for field-deployable diagnostics.
Discussion
In this study, we developed and systematically optimized a CRISPR-Cas13a-based detection platform for E. amylovora that operates without the need for nucleic acid amplification or RNA extraction. Our work demonstrates the feasibility of leveraging Cas13a collateral cleavage activity to achieve rapid, species-specific detection directly from plant tissues, a major step toward practical, field-deployable diagnostics for fire blight. By designing EA-specific crRNAs, screening candidates, and developing a robust alkaline lysis workflow compatible with plant matrices, this study addresses several challenges associated with CRISPR-based detection of bacterial plant pathogens.
A central challenge for plant-pathogen diagnostics is the presence of inhibitory compounds such as polyphenols, polysaccharides, oxidants, and secondary metabolites, that often interfere with enzymatic reactions (Kishor et al., 2020). Remarkably, the final extraction-free lysis workflow proved compatible with samples containing Malus domestica leaf tissue, enabling detection within 10 minutes and without RNA purification. This rapid performance is comparable to or faster than established CRISPR-based pathogen assays (Yang et al., 2023) and traditional molecular workflows that require DNA extraction and PCR amplification (Anonymous, 2022). Among the tested crRNAs, the V3 and recA547 guides consistently demonstrated high specificity and performance across purified synthetic RNA, native bacterial transcripts, and extraction-free plant suspensions. The redundancy provided by two validated guides enhances diagnostic reliability and provides flexibility when adapting the assay to different sample types or field conditions.
This study highlights several conceptual and technical insights relevant to the development of extraction-free CRISPR diagnostics. First, our results show that crRNA performance is strongly influenced by the biochemical environment of real samples; guides that appear highly effective against synthetic RNA can lose activity in crude lysates due to inhibitory components, altered RNA structures, or competition from background plant and bacterial RNA. These observations underscore that guide validation must be performed in matrix-relevant conditions, not solely with purified or synthetic targets. Second, our findings demonstrate that sample-driven interference is a major determinant of diagnostic success, often outweighing enzyme or probe optimization. Plant lysates introduce nucleases, secondary metabolites, and tissue-specific inhibitors that can trigger non-specific Cas13a activation or suppress true signal. Developing extraction-free assays therefore requires an integrated approach that simultaneously considers crRNA design, lysis chemistry, and the compositional complexity of the intended sample type. Together, these insights emphasize the need for diagnostics that are not only biochemically optimized but also environmentally resilient across diverse real-world matrices.
Validation with naturally infected field samples, where bacterial loads and matrix complexity can vary substantially, can further improve the presented detection system. While the platform reliably achieved rapid EA-specific detection of 1 × 106 CFUs/reaction within 15 minutes without nucleic acid purification or thermal cycling in the presence of plant material, the effective sensitivity in true field samples remains to be determined and may require further optimization for maximum sensitivity. Despite these considerations, the overall workflow provides many advantages such as minimal pipetting, compatibility with crude lysates, and no thermal cycling, and positions the system well for portable, low-infrastructure formats such as handheld fluorimeters or lateral flow devices. Together, these advancements lay essential groundwork for translating CRISPR-Cas13a diagnostics into practical, field-ready tools capable of strengthening plant disease surveillance and agricultural biosecurity.
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