Bar-cas12a, a Novel and Rapid Method for Plant Species Authentication: A Case of Phyllanthus Species

The rapid and accurate species diagnosis accelerates the performance to investigate various biology elds and its relevant, perhaps but morphology-based species taxonomy/identication hamper. DNA barcodes (Bar) has been employed extensively for plant species identication. Recently, CRISPR-cas system can be applied for diagnostic tool to detect pathogen’s DNA based on the collateral activity of cas12a or cas13. Here, we developed barcode-hyphenated with cas12a assay, “Bar-cas12a” for species authentication using Phyllanthus amarus as a model. The gRNAs were designed from trnL region, namely gRNA-A and gRNA-B. As a result, gRNA-A was highly specic to P. amarus amplied by RPA in contrast to gRNA-B even in contaminated condition. Apart from the large variation of gRNA-A binding in DNA target, cas12a- specic PAM’s gRNA-A as TTTN can be found only in P. amarus. PAM site may be recognized one of the potential regions for increasing specicity to authenticate species. In addition, the sensitivity of Bar-cas12a using both gRNAs gave the same detection limit at 0.8 fg and it was 1,000 times more sensitive compared to agarose gel electrophoresis. Overall, Bar-cas12a using trnL-designed gRNA offer a highly specic, sensitive, speed, and simple approach for plant species authentication and is likely to implement point-of-care testing.


Introduction
Species authentication/discrimination is an essential task in various areas in biology systematics, ecology, evolution, forensics, food science, medical as well as even herbal and cosmetic industries, leading to correct species exploitation regarding their purposes [1][2][3][4][5][6][7][8] . Traditional species taxonomy has been performed using the external morphological features or microanatomy which tightly requires the complete ower features or complete signi cant characteristics for species identi cation by an expert 9 . Perhaps, in several areas, the specimens obtained have been incomplete forms, immature stage, or modi ed/processed samples without key characters to identify, contributing to di culty/impossibility in species identi cation and hampering the advance of investigation or research 9 . In several decades ago, advanced molecular approaches e.g., hybridization, DNA ngerprint, DNA barcodes, high resolution melting (HRM) have been used widely and extensively for facilitating species authentication in various organisms 2-6, [10][11][12][13][14][15][16] . Certainly, these molecular approaches enable species identi cation despite the specimens with completely damaged but DNA existing, especially DNA barcodes (Bar) which there are many regions exhibiting a successful species discrimination for plant species (e.g., rbcL, matK, trnL, and ITS) [15][16] . However, they are relatively complex, time and cost-consuming because they require expensive equipment (e.g., thermal cycler, realtime PCR, sequencer machine).
Currently, nucleic acid isothermal ampli cation (e.g., RCA, LAMP and RPA) has been emerging and gaining attention for RNA/DNA ampli cation, in particular pathogen detection as they require only heat box or water bath, leading to adaptation for point-of-care testing [17][18][19] . RPA is one of isothermal ampli cation based on enzymatic activities relating to DNA replication process and the reaction can be performed at constant temperature in range of 30-45°C for DNA ampli cation (optimal temperature at 37°C) 18-19 , mycoplasma 20 , and virus/viroid RNA in plant 21 .
Recently, CRISPR-cas systems exhibited the high potential for genome editing with accuracy and precise in the speci c DNA target and included the adaptation for pathogen diagnostic with high sensitivity, speci city, simplicity, and speed, for instances, HPV-16 and 18 [22][23] and shrimp pathogens e.g., white spot syndrome virus (WSSV) 24 . The cas12a can be applied as diagnostic tool because it has the collateral activity or trans-activity for cleavage of non-target single stranded (ss) DNA once forming a tertiary complex (cas12a-gRNA-target) 25 . ssDNA is designed as reporter based on uorescence resonance energy transfer (FRET) between uorescence and its quencher or antigen-antibody interaction by lateral ow dipstick readout [22][23][24]26 .
Herein, we would like to establish a novel method for plant species authentication using the hyphenation of plant DNA barcode, trnL and cas12a, namely "Bar-cas12a". In this study, Phyllanthus species including Phyllanthus amarus, Phyllanthus urinaria, Phyllanthus debilis, Phyllanthus virgatus, were used as a model to validate Bar-cas12a for species authentication of P. amarus because they have similar morphological features and have been used as herbal commercialized products.

Condition optimization of cas12a assay
In this study, we presented two gRNAs designed from trnL region which were speci c to P. amarus. The gRNA-A were designed in the opposite direction to gRNA-B and the variation nucleotide in binding site of gRNA-A was more diverse than that of gRNA-B which existed only single point (Fig. 1A). Both of two gRNAs for cas12a assay were successfully produced by in vitro transcript which duplex DNAs for two gRNAs were used as the templates for gRNA synthesis as depicted in Fig. 1A and 1B. The scheme illustration for the principle of in vitro digestion of cas12a were shown in Fig. 1C. In addition, the concentration of cas12a and gRNA gave the highest uorescence from the cleavage of ssDNA reporter at 37°C for an hour was at 100 nM : 100 nM whereas there was no uorescence in control (without DNA target) (Fig. 1D).

Species authentication performance of Bar-cas12a
To evaluate the performance of Bar-cas12a assay for species authentication of P. amarus. For speci city determination, trnL region of different four Phyllanthus species were ampli ed by RPA using modi ed universal trnL primer, yielding about 600 bp ( Fig. 2A) and RPA products of four species were used for cas12a using gRNA-A and gRNA-B. Our results displayed that gRNA-A gave the uorescence signal with speci c PA while gRNA-B was positive uorescence signal for all species tested (Fig. 2B). In addition, the sensitivity assay was done using the different starting amount of DNA's P. amarus for DNA ampli cation by RPA in range of 0-80 ng. For RPA ampli cation under agarose gel electrophoresis, we found that the limit of detection (LOD) was at 0.8 ng which gave DNA band as depicted in Fig. 2C-D. Meanwhile Bar-cas12a using gRNA-A and gRNA-B was observed for positive uorescence signal in range of 0.8 pg − 80 ng for both gRNAs, indicating signi cantly more sensitivity than the agarose gel electrophoresisvisualized RPA assay ( Fig. 2C-D).
In addition, species authentication by Bar-cas12a was validated using the admixture between P. amarus and P. urinaria with different amount of DNA proportion. Indeed, all admixtures gave the positive for DNA ampli cation by RPA (Fig. 3A). However, Bar-cas12a with gRNA-A demonstrated greater capability of species authentication with high speci city than gRNA-B because Bar-cas12a with gRNA-B produced positive results (100%) for P. urinaria but not with gRNA-A ( Fig. 3B-E). Moreover, we found that Bar-cas12a using gRNA-A produced positive results even with a relatively small amount of P. amarus DNA (only 2%) in the admixture condition ( Fig. 3B-C).
Discussion CRISPR-cas system has not been only accomplished for genome editing in the certain target for various organisms but it can also be adapted for e cient diagnostic tool with high sensitivity and speci city to detect the pathogens [20][21][22][23][24]26 . In addition, this approach offers high potential for applying point-of-care testing as of short-time process without sophisticated equipment [20][21][22][23][24]26 . This was the rst report to apparently exhibit the feasibility of hyphenating DNA barcode and cas12a assay to authenticate plant species as P. amarus. Our signi cant ndings demonstrated that Bar-cas12a using gRNA-A of trnL barcode based on RPA enabled to speci cally authenticate P. amarus with high sensitivity of LOD at 0.8 fg which was three orders of magnitude more sensitive than RPA visualized by agarose gel electrophoresis. Additionally, Bar-cas12a using gRNA-A enables species authentication of P. amarus even after contaminated with P. urinaria.
In the present study, gRNA-A and gRNA-B were designed based on the trnL sequences derived from the different four species including P. amarus, P. urinaria, P. debilis and P. virgatus because of the high variation sequences [27][28] . Although all of them share common morphological features, P. amarus is a single species existing bioactive compound of phyllanthin and hypophyllathin responsible for hepatoprotection 29 . P. amarus has been commercialized as various product forms e.g., tea infusion, capsule, and tablets 6 . Thus, P. amarus was employed as a plant models to authenticate by Bar-cas12a assay. For cas12a condition, the su cient and suitable concentration ratio of cas12a and gRNA to form binary complex and trigger the activity for positive uorescence within an hour was 100 nM: 100 nM.
Several reports revealed that the optimal concentration between cas12a and gRNA for detecting the target were used in various concentrations and ratios from 200 nM : 500 nM 20 to the least at 30 nM : 36 The speci city and sensitivity of Bar-cas12a using either gRNA-A or gRNA-B for P. amarus authentication were determined. Obviously, the speci city of gRNA-A was the P. amarusspeci c marker due to other species without the positive uorescence whereas gRNA-B gave the positive for all species. Furthermore, we veri ed the species authentication ability of Bar-cas12a in contaminated conditions with unwanted species by admixing different amounts of P. amarus and P. urinaria DNA. Our ndings demonstrated that Bar-cas12a using gRNA-A is highly species-speci c to P. amarus rather than using gRNA-B. With these regards, it was unsurprise that Bar-cas12a using gRNA-A have more speci city than gRNA-B as the gRNA-A was designed based on PAM site of cas12a (TTTG) in front of the DNA target which it existed only in P. amarus while the other species without PAM sites because they are TCTG. In contrast, gRNA-B contain only a single variable site although a variable site (A > G) was in the seed regions (1-5 rst base next to PAM site) 25 . This might suggest that the presence of a single variable site at seed region may be insu cient to distinguish the different nucleotides or SNP within DNA target. The variation of PAM site might be one of the e cient targets to discriminate difference in nucleotide for SNP genotyping or species discrimination/authentication. For sensitivity assay, Bar-cas12a using both gRNA-A and gRNA-B provide the LOD at 0.8 fg which was three orders of magnitude more sensitive than agarose gel electrophoresis-visualized RPA. This indicated that the hyphenation of cas12a enable to increase the sensitivity via the signal ampli cation of DNase activity of cas12a triggered by speci c DNA target. Cas12a coupled with nucleic acid ampli cation such as LAMP or RPA has been achieved for detecting plant RNA viruses 21 , HPV16 and HPV18 [22][23] and bacterial contamination in food 30 , with high speci city and sensitivity.
Here, we describe the feasibility of implementing cas12a combined with isothermal ampli cation to facilitate species authentication of P. amarus. However, this approach still having inherent limitation for being highly speci c, sensitive, speed and simple tool for diagnostics is that it requires an ampli cation process to increase a large amount of DNA target to activate the collateral activity of cas12a. Given that direct DNA extracts can be used as template to be performed by cas12a without DNA ampli cation, we strongly believe that the approach would be near the ideal method for rapidity. Hence, we purpose a concept to reduce DNA ampli cation step by use of the multiple gRNAs to bind in different but speci c DNA target, contributing to increasing amount of activated cas12a in the reaction.
In summary, our ndings demonstrated that Bar-cas12a serve as immensely promising tool with highly speci city, sensitivity, speed, and simplicity for species discrimination/authentication in plant species especially in genus Phyllanthus. We proposed that this approach is a new shed of light in accommodating species discrimination/authentication for point-of-care testing which make us identify or distinguish plant species/commercial product in elds without the sophisticated equipment in two hours.

Specimens and DNA extraction
Phyllanthus species including P. amarus, P. urinaria, P. debilis and P. virgatus were collected around Naresuan University, Phitsanulok, Thailand and these species were identi ed through a key from Flora of Thailand Euphorbiaceae (http://www.nationaalherbarium.nl/ThaiEuph/ThPspecies/ThPhyllanthusT.htm). The experiment and collection of plants complied with guidelines of Department of Biology, Faculty of Sciences, Naresuan University. In this study, P. amarus was used a plant model to authenticate by cas12a assay because it has been extensively used for medical purpose. Leaves were used for DNA extraction by Genomic DNA isolation kit (PureDireX, Taiwan). The quality and quantity of DNA obtained were measured by Nanodrop (Thermo Scienti c, USA) and 1% agarose gel electrophoresis. DNA were diluted as 20 ng/ul and stored at -20°C for further use.
Design and synthesis of guide RNA for cas12a To generate suitable gRNAs for cas12a assay, trnL region of the four species was conducted for multiple alignment by MEGA X. There were two signi cant points as guideline for gRNA design to species differentiation: (i) searching for protospacer adjacent motif (TTTV (V = A, G or C)) and (ii) the sequences for DNA targets having variation among four species in the seed sequences (1-5 rst bases next to PAM) 25 , given as gRNA-A and gRNA-B for speci c P. amarus (Fig. 1A). The synthesis of gRNA was done by in vitro transcription (IVT) under double stranded (ds) DNA as a template. The dsDNA was constructed and synthesized from Integrated DNA technologies (IDT, USA) which consisted of three parts as (i) T7 promoter regions, (ii) tracrRNA to incorporate with cas12a to form binary complex and (iii) crRNA to bind with DNA target, forming a tertiary complex. These dsDNAs were used as template for RNA synthesis via in vitro transcription (IVT) by HiScribe™ T7 Quick (#E2050S, NEB, US). The synthetic gRNAs were puri ed to remove the impurities by the Monarch RNA Cleanup Kit (50 µg) (NEB, US). The synthetic gRNA products were measured for amount and purity by Nanodrop and 2% agarose gel electrophoresis and then adjusted for concentration as 10 µM for further study.
In vitro cas12a assay In this experiment, the concentration ratio of cas12a or cpf1 (#M0653T, NEB, US) and gRNA were varied to nd out the suitable condition of in vitro digestion of cas12a. The ratio of cas12a and gRNA was constantly done at 1 : 1 but the concentrations were varied from 12.5 nM : 12.5 nM to 100 nM : 100 nM. Firstly, the binary complex between cas12a (cpf1) and gRNA was formed under admixture of 1X 2.1 NEB buffer, 100 nM cpf1, 100 nM gRNA-A or -B and then incubated at 37°C for 10 minutes. Subsequently, 1 µl of 50 µM single stranded DNA reporter (ssDNA reporter) (FAM/TTATT/3IABkFQ) and 5 µl of DNA targets (RPA products) were added. Finally, the nuclease-free water was added to 24 µl and incubated at 37°C for an hour. The cleavage of ssDNA report was determined under LED transilluminator to visualize the orescence signal by visible eye.
Speci city and sensitivity determination of Bar-cas12a assay To assess the speci city of cas12a assay for species authentication of P. amarus, gRNA-A and gRNA-B were compared by PCR products which were ampli ed from DNA's different Phyllanthus species including P. amarus, P. urinaria, P. debilis and P. virgatus. DNA ampli cation of these species were performed by RPA. A reaction of 25-µl volume were consisted of 1X reaction buffer, 1X probe E-mix, 1.8 mM, 0.48 µM forward primer (CGAAATCGGTAGACGCTACG), 0.48 µM reverse primer (GGGGATAGAGGGACTTGAAC), 1X core reaction mix, 20 ng of DNA template or plant extract and 14 mM magnesium acetate on the lid of PCR tube and added nuclease free water to 25 µl. The reaction was performed under heating box at 37°C for 40 minutes. The RPA products were checked by 1.5% agarose gel electrophoresis. Afterward, RPA products of different species ampli ed were used as DNA targets for assessing the speci city of Bar-cas12 assay using gRNA-A or gRNA-B.
To evaluate the sensitivity of Bar-cas12a assay using gRNA-A and gRNA-B, the diluted DNA's P. amarus were varied for 0.8 fg to 80 ng to amplify by RPA. Subsequently, RPA products were detected for P. amarus by Bar-cas12a. The uorescence which indicates the presence of DNA target was recorded every minute for 2 hours of incubation under realtime PCR and after completing the reaction of cas12a assay, the tubes was determined under LED transilluminator to visualize the orescence signal by visible eyes.

Species authentication by Bar-cas12a
To test the species authentication performance of Bar-cas12a assay using gRNA-A and gRNA-B, the admixture between two species of P. amarus and P. urinaria was done in different amount percentage proportion, given as 100%:0%, 50%:50%, 25%:75%, 10%:90%, 2%:98% and 0%:100%, respectively. The initial DNA concentration of each species used to create an admixture was 10 ng/µl. Subsequently, these admixtures were employed to authenticate P. amarus by the Bar-cas12a using gRNA-A and gRNA-B with realtime PCR to acquire the uorescence signal over time, and then PCR tubes were observed under LED transilluminator. Figure 1 The  by PCR and Bar-cas12a using gRNA-A and gRNA-B and the uorescence signal were monitored for cleavage of ssDNA reporters by realtime PCR within two hours. Raw gures of agarose gel electrophoresis for speci city and sensitivity were in Figure S2 and S3, respectively.