1 Abstract

Experiencing a loss of a relative to advanced breast cancer, a member of our iGEM team brought up the idea of developing a diagnostic tool for early stage breast cancer so that such tragedy might be avoided.  Breast cancer has become the most common cancer in the world with the highest mortality in female cancers in 2020. Early diagnosis of breast cancer and individualized optimization of treatment strategies can greatly improve the survival. Biomarkers such as circRNAs are useful in the screening, diagnosis, prognosis and drug resistance prediction of breast cancer and are important for making treatment decisions. Our team aims to find a practical way to sensitively detect circRNA biomarkers for breast cancer. The current detection methods for circRNAs often require laboratory equipment and high cost. Our project offers a sensitive, cheap, noninvasive, and widely applicable alternative way for sensing circRNA biomarkers for breast cancer. Our detection tool combines a CRISPR-Cas12a technology with a cell-free system to identify two circRNA biomarkers, hsa_circ_0001785 and hsa_circ_0001982, for the diagnosis and prognosis of breast cancer patients. The detection is sensitive for minimally invasive samples like blood and can be further extended to other circRNA biomarkers with ease.

2 Breast cancer and biomarkers

Accoding to the GLOBOCAN 2020 database, there were 2.3 million new breast cancer patients in 2020 surpassing lung cancer to be the most prevalent cancer in the world. Among all female cancers, breast cancer has both the top incidence (24.2%) and the top mortality (15.5%) in 2020 (Sung, 2021).

A

B

Figure 1 Distribution of cases and deaths for the top 10 most common cancers in 2020 for (A) both sexes, and (B) women. Source: GLOBOCAN 2020 (Sung, 2021)

Treatment selection for breast cancer is generally based on clinical stages and molecular subtypes. Stage I-II patients are usually treated with surgery and radiotherapy and have a 5-year survival rate reaching 90%, while stage IV patients are mainly treated with systemic drug therapies and have a 5-year survival rate around 20% (DeSantis, 2019).  Breast cancer is heterogeneous with varying clinical presentations and treatment responses, due to genetic, epigenetic and transcriptome differences among patients. Identification of subtypes based on various biomarkers is important for predicting treatment response and prognosis. The expression of ER, PR and HER2 is used to broadly classify breast cancer into 4 molecular subtypes. ER and PR expression correlates with tumor response to hormone therapy and good prognosis. Patients with HER2 expression respond to HER2 targeted drugs, and have unfavorable prognosis. Patients absent of ER, PR and HER2 expression are usually treated with immunotherapy and chemotherapy and have worst prognosis. Other molecular biomarkers are used to further divide these subtypes into subgroups (Eliyatkin, 2015).

Therefore, screening biomarkers for early diagnosis can improve survival, and biomarkers for molecular subtype diagnosis, treatment response, drug resistance prediction and prognosis are valuable for optimizing customized treatment strategies for breast cancer patients to achieve better survival and quality of life.

3 circRNAs as biomarkers for breast cancer 

circRNAs are a class of non-coding RNAs. Many circular RNAs (circRNAs) are found to be associated with clinical stage, molecular subtype, treatment response, drug resistance and prognosis of breast cancer, and a large number of them are biomarkers for early diagnosis, diagnosis of clinical stages and molecular subtypes, and prediction of drug resistance and prognosis. For example, the expression of hsa_circ_1785 is increased in the serum of breast cancer patients and is positively correlated with tumor histological grade and TNM stage (Yin, 2017). circDENND4C expressed in breast cancer tissues is proportional with tumor size (Liang, 2017). These circRNAs could be biomarkers for clinical-stage diagnosis. hsa_circ_104689 and hsa_cirC_104821 are highly expressed in PR-negative invasive ductal carcinoma tissues of breast, while hsa_circ_406697 was lowly expressed (Lü, 2017). circRNAs that silence the function of Raf can affect drug resistance to chemotherapy drugs of breast cancer via mitogen-activated protein kinase (MAPK) regulation (Gao, 2017). circFOXO3 is closely associated with the prognosis of breast cancer by targeting a tumor suppressor gene, Forkhead box O3 (FOXO3) (Smit, 2016).

circRNAs have several advantages for biomarker detection:

(1)Easy sampling

circRNAs are found in the tissue, blood, urine, gastric juice and breast milk of breast cancer patients. Tissue sampling needs invasive operations like surgery and biopsy. circRNA detection can use blood and urine samples by minimally invasive procedures. The easy sampling reduces complications and results in better compliance of patients.

(2)Stability

circRNAs are characterized by covalent closed-loop structure, so they are less degradable and more stable than linear RNAs. This stability facilitates the detection of circRNAs. 

(3)Universality

Many circRNAs associated with genomes exist in various organisms, such as humans, mice, zebrafish, nematodes and fruit flies, etc., and their traces have even been found in blood, saliva and exosomes. This ensures that our work and future research can be based on entity experiments, which is beneficial to clinic test and further popularization.

4 Detection methods for circRNAs

Current detection methods for circRNAs involves RNA-sequencing (RNA-seq), microarray techniques, RT-qPCR, Digital Droplet PCR (ddPCR), Northern blotting, NanoString nCounter and etc.  

4.1 RNA-seq

RNA-seq is a transcriptome sequencing technology, and samples are sequenced using high-throughput sequencing technology.  Its advantages are digital signal, high sensitivity and whole genome analysis.  But RNA-seq detects total RNA, which limits the number and accuracy of other RNAs since a large proportion of RNA in cells comes from ribosomes and mitochondria.  RNA-seq is too expensive to start up for the average laboratory, and the analyses require ample computational power and bioinformatics expertise (Kristensen, 2019).

4.2 Microarray technology

Microarray technology is a general laboratory method that uses arrays of thousands to millions of known nucleic acid fragments combined on a solid surface to detect DNA or RNA sequences isolated from samples through hybridization. It can simultaneously analyze a large number of circRNAs. But the cost of single experiment is high, the specificity and accuracy are medium, and the experimental equipment is highly sensitive to the hybridization temperature, the purity and degradation rate of genetic material, and the amplification process (Chua, 2003).   

4.3 RT-qPCR

RT-qPCR reversely transcribes circRNAs into cDNA, and amplifies cDNA with specified polymerized primers for sequence detection. It can provide sensitive detection with quantitative data. But the rolling circle amplification could compromise its accuracy. RT-qPCR requires expensive laboratory equipment and the experiment cost is medium (Kristensen, 2019).

4.4 ddPCR

ddPCR combines microfluidics technology and TaqMan-based PCR to detect and quantify DNA and RNA with sensitivity and specificity. It is simpler, faster, and more accurate than real-time PCR and can work with smaller samples (Mazaika, 2014). ddPCR requires specialized equipment and the experiment cost is high.

4.5 Northern blotting

Northern blotting is shown to be the gold standard for circRNA analysis, which strongly indicates the presumed circRNA ring structure.  Strictly speaking, circRNA validation usually requires Northern blotting in combination with other tools such as RNase R and RNase H treatments.  Though the experiment cost is relatively low, the sensitivity and accuracy are low and it is labor intensive (Kristensen, 2019). 

4.6 NanoString nCounter

NanoString nCounter is a relatively new multiplex nucleic acid hybridization technology that can assess hundreds of genes in a dozen samples in one assay with high sensitivity and accuracy and give digital quantification. The technique works well with a variety of sample types, requires small RNA input and no enzymatic reactions (Goytain, 2020). Recent studies have found that NanoString nCounter can be used to detect circRNAs in high and low quality RNA samples. Yet, this technique requires specialized equipment and the experiment cost is high (Kristensen, 2019). 

Figure 3 The equipment of NanoString nCounter (www.yiqi.com/pt7155/list03_1_0_1571.html)

Table 1 Methods for detecting and quantifying circRNA (Kristensen, 2019)

5 Our Project

Our project aims to develop an economical and effective approach to detect circRNA biomarkers in the various samples of breast cancer patients, in order to better predict the diagnosis, prognosis, and therapy resistance of breast cancer patients and help physicians to make better-individualized treatment decisions for the patients.

     

Our circRNA detection platform couples a CRISPR-Cas12a system with a cell-free transcription-translation (TXTL) system. Due to the igRNA in the CRSPR-Cas12a system that can specifically recognize the target circRNA, the tool has high specificity. In addition, our tool can be performed with ease and convenience. To use the detection platform, an RNA sample, extracted from a human sample such as tissue or blood, is added into a 1.5mL microcentrifuge tube containing all reagents required. The presence of the targeted circRNA can be identified by the CRISPR-Cas12a system within the tube at room temperature and a fluorescence signal can be detected under UV light. The whole process lasts only about 15min. No specialized equipment or professional specialists are required and the cost for each experiment is low.  

6 Project design

Our project combined the CRISPR-Cas12a technique with a cell-free system to sensitively detect two circRNAs, hsa_circ_0001785 and hsa_circ_0001982, in the blood samples of breast cancer patients.   The detection of hsa_circ_0001785 and hsa_circ_0001982 can be used for the early diagnosis of breast cancer and hsa_circ_0001982 is also a biomarker for the poor prognosis of TNBC patients with conventional chemotherapy.

6.1 Overall design

Our tool is a cell free system containing all molecular machineries needed for the CRISPR-Cas12a-based detection process. The CRISPR-Cas12a system in our tool mainly consists an igRNA, Cas12a enzymes, double strand DNA (dsDNA) substrate and a reporter DNA (fluorescence probe) which is a single strand DNA (ssDNA). The igRNA is specifically designed to recognize the trigger RNA (trRNA) sequence that is unique of the target circRNA. After the recognition, the igRNA is activated and subsequently bind to the dsDNA. This binding can activate Cas12a enzyme and make the enzyme to cleave DNAs in the system, including the reporter DNA. The cleavage of the reporter DNA separates a quencher dye gene BQ1 away from a reporter dye gene FAM, so that the fluorescence signal of FITC protein expressed by the FAM gene is emitted, which can be detected under UV light and analyzed by image J software.

A: igRNA in its inactive state; B: Activation of igRNA after hybridization with the trRNA; C: igRNA activation causes igRNA bind with the dsDNA and thus activates Cas12a enzyme, leading to random cleavage of DNAs including the reporter DNA by Cas12a enzyme, resulting in the separation of FAM (a reporter dye) and BQ1 (a quench dye) and unquenching of FITC fluorescence

The guide (red) sequence is complementary to the dsDNA; VR1 and VR2 sequences can bind to the trRNA (VR1’+VR2’) of the target circRNA; The black part is a Cas12 handle that binds with Cas 12a enzyme.

Figure 4 Schematic representation of the CRISPR-Cas12a system in our project

6.2 Detailed design

We developed our circRNA detection tool for breast cancer by completing the following missions.

(1) Selection of target circRNAs

Target circRNAs for detection are carefully selected according to the following two criteria: (1) target circRNAs should be proven previously to be associated with the genesis and development of breast cancer and could be appropriate biomarkers for screening, diagnosis, drug resistance or prognosis. (2) target circRNAs should have a favorable secondary structure for high specificity of the CRISPR-Cas12a system.

In this project, we selected hsa_circ_0001785 and hsa_circ_0001982 as biomarkers for the early diagnosis of breast cancer and hsa_circ_0001982 can also be used as a biomarker for the poor prognosis of TNBC patients with conventional chemotherapy. Previous studies have shown that hsa_circ_0001785 inhibits the proliferation, migration and invasion of breast cancer cells in vivo and in vitro (Li, 2020), is aberrantly expressed in the peripheral blood of breast cancer patients and is a stable biomarker for the diagnosis and progress of breast cancer (Yin, 2018). hsa_circ_0001982 is overexpressed in breast cancer tissue and cell lines. Its inhibition suppresses the proliferation and invasion, and induces the apoptosis of breast cancer cells (Tang, 2017). Silencing of hsa_circ_0001982 undermines multiple drug resistance (MDR) of the drug-resistant TNBC cell line. TNBC patients with decreased serum level of hsa_circ_0001982 are less likely to relapse after conventional chemotherapy. It is an indicator of poor survival of TNBC patients (Li, 2021). Both hsa_circ_0001785 and hsa_circ_0001982 have suitable secondary structure for high specificity of the CRISPR-Cas12a system.

Figure 5 The secondary structure of hsa_circ_0001785 (http://biophy.hust.edu.cn/new/3dRPC/create)

Figure 6 The secondary structure of has_circ_0001982(http://biophy.hust.edu.cn/new/3dRPC/create)

(2) CRISPR-Cas12a system

Clustered regularly interspaced short palindromic repeats (CRISPR) is a family of DNA sequences found in the genomes of prokaryotic organisms. CRISPR, along with Cas proteins, is acquired immune system in archaea and bacteria that can identify previously invaded foreign DNA sequences and break them (Chen, 2018). Due to its specificity and convenience, CRISPR-Cas system is widely adopted to detect nucleic acids (Leung, 2021). In an engineered CRISPR-Cas system, a single-guided RNA (sgRNA) binds to a target DNA and is thus activated to bind with a nuclease, Cas enzyme, resulting in the cleavage of the target DNA.

The CRISPR-Cas12a system used in our detection tool contains an RNA-interacting guide RNA (igRNA), Cas12a enzyme, dsDNA and a reporter DNA (fluorescence probe).

A: igRNA

We engineered the igRNA to detect a unique RNA sequence (named trigger RNA, trRNA) of the target circRNA and subsequently activate Cas12a enzyme to randomly cut DNAs including the reporter DNA in the system.

Our igRNA mainly contains the following parts:

i) Cas12a handle

Cas12a handle is a conserved sequence of sgRNA that binds with the Cas12a enzyme.

ii) Guide

The guide is a sequence that can bind with dsDNA and then activate Cas12a enzyme. dsDNA is added into the system abundantly so that the activation of the enzyme is always strong.

iii) Trigger (VR1+VR2)

Trigger is designed to specifically bind with the trRNA. trRNA is a unique sequence of the target circRNA, so the trigger can recognize the circRNA with high specificity. The trigger is separated by a clamp, so it contains two separate sequences, named VR1 and VR2.

iv) Clamp

Clamp splits the trigger and is complementary to a portion of the guide (Figure 7; Galizi R, 2020). Clamp blocks the guide, so the guide cannot bind to the dsDNA.

When the target circRNA is in the sample, the trigger hybridizes with the trRNA, hence the clamp unblocks the guide (Figure 4). The guide then binds with the dsDNA, which activates Cas12a enzyme, resulting in the cleavage of DNAs including the reporter DNA (fluorescence probe) in the system.

In conclusion, the igRNA contains the following three components: Cas12a handle, guide of igRNA, and trigger RNA-sensor (trigger+clamp) of igRNA. Figure 7 illustrates the structure of igRNA. In this experiment, we designed different sequences for igRNA to fit into experimental needs and compared the fluorescence produced by each system using different igRNA and dsDNA combinations.

Figure 7: Illustration of the inactivated igRNA

We designed the following parts in our project:

BBa_K4409006, BBa_K4409007, and BBa_K4409008 are dsDNAs based on the sequences of GAPDH, hsa_circ_0001785 and hsa_circ_0001982, respectively. BBa_K4409001 and BBa_K4409002 are guide sequences of igRNA complimentary to BBa_K4409007, and BBa_K4409008.  BBa_K4409003, BBa_K4409004, and BBa_K4409005 are serving as trigger-RNA sensors of igRNA. These three sequences are also based on hsa_circ_0001982 and hsa_circ_0001785.Therefore, they can detect our target circRNAs.

In addition, we constructed different composite parts to form igRNA/gRNA serving various functions. Here is a table to illustrate the components of different composite parts we designed.

Table 2 Composite part list

Name of parts	Cas12a handle	Guide	TriggerRNA sensor
BBa_K4409009	AAG sequence	GAPDH as guide	Based on hsa_circ_0001982
BBa_K4409010	CT sequence	GAPDH as guide	Based on hsa_circ_0001982
BBa_K4409011	CT sequence	hsa_circ_0001785 as guide	Based on hsa_circ_0001982
BBa_K4409012	CT sequence	hsa_circ_0001982 as guide	Based on hsa_circ_0001785
BBa_K4409013	CT sequence	GAPDH as guide	Absent (Negative control)
BBa_K4409014	CT sequence	Based on BBa_K3859000	Absent (Negative control)
BBa_K4409015	AAG sequence	Based on BBa_K3859000	Absent (Negative control)
BBa_K4409016	AAG sequence	GAPDH as guide	Absent (Negative control)

The main function of those bio-bricks is serving igRNA function. Later by comparing the effects of these biobricks, we can get an understanding of the efficiency of different bio-bricks and choose the most appropriate one for our system. We later expressed the igRNAs/gRNAs in plasmids and compared the fluorescence produced by these igRNAs/gRNAs.

Click here for specific details for different parts utilized in this experiment.

B: Cas12a enzyme

Among different variants of Cas enzymes, Cas9, Cas12a, and Cas13 are the most widely-used proteins for CRISPR-based diagnostics (Kaminski, 2021).  In this experiment, we chose to adopt Cas12a enzyme, due to its cheaper probes and fewer steps required. Cas12a enzyme has a number of applications in the disease detection realm, such as the detection of SARS-CoV-2 as well as human papillomavirus (Broughton, 2020).

Unlike Cas9, Cas12a employs a multistep quality control mechanism to ensure the accurate and precise recognition of target sequences. Unlike Cas13, Cas12a only cleaves double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA), so the plasmids and circRNAs in the system are prevented from being cleaved by enzymes.

Figure 8 Schematics of nuclease activities of Cas12a to cleave targeted DNA and non-targeted ssDNA. (Zhang, 2020)

C: Reporter DNA (Fluorescence probe)

A reporter DNA is a ssDNA used as a probe to give a visual signal when the CRISPR-Cas12a system is activated by the target circRNA. One side of the probe is an FAM gene and the other side is a BQ1 gene. A DNA sequence between the FAM gene and BQ1 gene is selected as a target DNA for our CRISPR-Cas12a system. FAM expresses a reporter dye, fluorescein isothiocyanate (FITC), which can emit fluorescence. BQ1 expresses a quencher dye. When the probe is intact, the close proximity of the quencher dye to FITC causes an energy transfer between the two dyes and suppresses the fluorescence of FITC. When the CRISPR-Cas12a system is activated, Cas12a enzyme breaks the reporter DNA, the two dyes are no longer in proximity and a fluorescent signal is observed.

Figure 9 Separation of FAM and BQ1 genes results in the emission of fluorescence

(3) Fluorescence detection

The peak for light absorption is 395 nanometers for FITC, so UV light is used to detect the fluorescence emitted by FITC. We designed a device with a UV lamp to observe the detection results in the most convenient and safest way. The device is consisted of a box, a sliding shelf holding eight-strip tubes, a UV lamp, and a window for observation.

(4) A cell-free transcription-translation system

A cell-free transcription-translation (TXTL) system manufactures target proteins and RNAs quickly from genes of interest via transcription and translation in vitro and can construct a biosensing platform. The system can be made with a crude cell lysate, or with purified elements of ribosomes, transfer RNA, enzymes, amino acids, energy supply systems, inorganic ions and other supplements to master a maximum control over the transcription and translation process (Copeland, 2021). It offers a way to easily modify reaction conditions, reduce the possibility of product toxicity and cut processing time and volume (Katzen, 2005).

In the cell-free system of our project, all reactions take place in a 1.5mL microcentrifuge tube, with a total volume of 20. Except igRNA, all of our elements, including fluorescence probe and Cas12a protein, come from an existing testing kit produced by a biotech company.

igRNA is expressed in vitro by adding 1 constructed plasmid, 1 p70 polymerase, 1 inducer (-galactoside, inducer of lac operon), and 9 LS70. The plasmid pET28a is selected as the expression vector for igRNA in our project. Plasmid pET28a contains T7 promoter and restriction enzyme cutting sites, XbaI and XhoI. These elements play important role in the reconstruction and optimization of plasmids and in the expression of igRNA in the cell-free system. T7 polymerase is also vital in this system.

Figure 10 Genetic circuit of the plasmids for igRNA expression in our project

   

To sum up, the overall structure of our system is: target circRNA activates igRNA; igRNA activates Cas12a protein; dsDNA substrate put Cas12a protein in secondary activation stage; the reporter ssDNA is cut; and finally the fluorescence signal appears.

References 

Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians, 71:209–249(2021).

DeSantis CE, Ma J, Gaudet MM, et al. Breast cancer statistics, 2019. CA: A Cancer Journal for Clinicians, 69(6): 438-451(2019).

Eliyatkın N, Yalçın E, Zengel B, Aktaş S, Vardar E. Molecular classification of breast carcinoma: from traditional, old-fashioned way to a new age, and a new way. Journal of Breast Health, 11(2):59-66(2015).

Valachis A, Carlqvist P, Ma Y, et al. Overall survival of patients with metastatic breast cancer in Sweden: a nationwide study. British Journal of Cancer, (2022).

Yin W, Yan M, Fang X, et al. Circulating circular RNA hsa_circ_0001785 acts as a diagnostic biomarker for breast cancer detection. Clinica Chimica Acta, (2017).

Liang G, Liu Z, Tan L, et al. HIF1alpha-associated circDENND4C promotes proliferation of breast cancer cells in hypoxic environment. Anticancer Research, 37(8):4337-4343(2017).

Lü L, Sun J, Shi P, et al. Identification of circular RNAs as a promising new class of diagnostic biomarkers for human breast cancer. Oncotarget, 8(27):44096-44107(2017).

Gao D, Zhang X, Liu B, et al. Screening circular RNA related to chemotherapeutic resistance in breast cancer. Epigenomics, 9(9):1175-1188(2017).

Smit L, Berns K, Spence K, et al. An integrated genomic approach identifies that the PI3K/AKT/FOXO pathway is involved in breast cancer tumor initiation. Oncotarget, 7(3):2596-2610(2016).

Kristensen LS, Andersen MS, Stagsted LVW, et al. The biogenesis, biology and characterization of circular RNAs. Nature Reviews Genetics, 20:675–691(2019).

Chua MS,Mansfield E,Sarwal M. Applications of microarrays to renal transplantation: progress and possibilities. Frontiers in bioscience, s913-23(2003).

Mazaika E, Homsy J. Digital droplet PCR: CNV analysis and other applications. Current Protocols in Human Genetics, 82:7.24.1-13(2014).

Goytain A, Ng T. NanoString nCounter Technology: High-throughput RNA validation. Methods in Molecular Biology, 2079:125-139(2020).

Li Z, Zheng J, Lin W, et al. Circular RNA hsa_circ_0001785 inhibits the proliferation, migration and invasion of breast cancer cells in vitro and in vivo by sponging miR-942 to upregulate SOCS3. Cell Cycle, 19(21): 2811-2825(2020).

Yin WB, Yan MG, Fang X, et al. Circulating circular RNA hsa_circ_0001785 acts as a diagnostic biomarker for breast cancer detection. Clinica chimica acta, 487: 363-368(2018).

Tang YY, Zhao P, Zou TN, et al. Circular RNA hsa_circ_0001982 promotes breast cancer cell carcinogenesis through decreasing miR-143. DNA and cell biology, 36(11): 901-908(2017).

Li H, Xia Z, Liu L, et al. Astragalus IV undermines multi-drug resistance and glycolysis of MDA-MB-231/ADR cell line by depressing hsa_circ_0001982-miR-206/miR-613 Axis. Cancer Management and Research, 13: 5821(2021).

Copeland CE, Langlois A, Kim J, et al. The cell-free system: A new apparatus for affordable, sensitive, and portable healthcare. Biochemical Engineering Journal, 175: 108124(2021).

Katzen F, Chang G, Kudlicki W. The past, present and future of cell-free protein synthesis. Trends in Biotechnology, 23(3): 150-156(2005).

Lyu Y, Peng R, Liu H. Protocells programmed through artificial reaction networks. Chemical Science, 11, 631-642(2020).

Chen JS, Ma E, Harrington LB, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360(6387): 436-439(2018).

Leung RKK, Cheng QX, Wu ZL, et al. CRISPR-Cas12-based nucleic acids detection systems. Methods(2021).

Galizi R, Duncan JN, Rostain W, et al. Engineered RNA-interacting CRISPR guide RNAs for genetic sensing and diagnostics. The CRISPR Journal, 3(5): 398-408(2020).

Kaminski M M, Abudayyeh O O, Gootenberg J S, et al. CRISPR-based diagnostics. Nature Biomedical Engineering, 5(7): 643-656(2021).

Broughton JP, Deng X, Yu G, et al. CRISPR–Cas12-based detection of SARS-CoV-2. Nature Biotechnology, 38(7): 870-874(2020).

Zhang YM, Zhang Y, Xie K. Evaluation of CRISPR/Cas12a-based DNA detection for fast pathogen diagnosis and GMO test in rice. Mol Breeding, 40: 11 (2020).