The CRISPR–Cas12a system is widely used in nucleic acid detection and biosensing due to its high sensitivity, selectivity, and simple design. However, traditional CRISPR–Cas12a sensors, which rely on linear activators, face challenges such as limited operability and low stability. This study explored the impact of three different activator topologies—linear, planar, and steric—on the trans-cleavage activity of Cas12a. We developed a Cas12a-based switch using a planar activator, which demonstrated superior operability and maintained higher activity compared to linear activators. Using this planar activator, we achieved highly sensitive detection of hypochlorous acid, with a detection limit as low as 88 nM, outperforming chemical probe-based methods. The introduction of topological activators will open new avenues for the development of CRISPR–Cas12a-based biosensors, offering broad potential for diverse applications.

With the widespread cultivation of genetically modified (GM) crops, many countries have called for quantitative labeling of GM products. While recent advancements, such as CRISPR-based detection technologies have facilitated rapid on-site detection of genetically modified organisms (GMOs), these technologies face challenges in achieving absolute quantitative detection. Here, we developed a practical and simplified integrated vortexing-driven droplet digital CRISPR/Cas13a (VTD-CRISPR) detection platform, enabling absolute quantification of GM products. The VTD-CRISPR platform offers a rapid and convenient method for partitioning T7-RPA-CRISPR/Cas13a reaction mixtures, generating tens of thousands of uniform picoliter-scale polydisperse droplets within seconds through quick vortexing, and eliminating the need for sophisticated microfluidic chips or specialized equipment. We further demonstrated the high specificity and strong sensitivity of the VTD-CRISPR platform, enabling on-site rapid qualitative and absolute quantitative detection through visual analyses. Moreover, the VTD-CRISPR assay showed strong alignment with the droplet digital PCR-based method for precise quantitative detection of GM concentrations in real-world samples across a wide genomic range. Interestingly, we also validated a hand-shaking-driven droplet digital CRISPR assay, which demonstrated good performance and may offer greater potential applications for our assay. Overall, considering the time, cost, and convenience, VTD-CRISPR holds great promise for practical GMO quantitative detection applications.

The new generation of gene editing technologies, primarily based on CRISPR/Cas9 and its derivatives, allows for more precise editing of organisms. However, when the editing efficiency is low, only a small fraction of gene fragments is edited, leaving behind minimal traces and making it difficult to detect and evaluate the editing effects. Although a series of technologies and methods have been developed, they lack the ability for precise quantification and quantitative analysis of these products. Digital polymerase chain reaction (dPCR) offers advantages such as high precision and sensitivity, making it suitable for absolute quantification of nucleic acid samples. In the present study, we developed a novel platform for precise quantification of gene editing products based on microfluidic chip-based dPCR. The results indicated that our assay accurately identified different types of edited samples within a variety of different types, including more complex genomic crops such as tetraploid rapeseed and soybean (highly repetitive sequence). The sensitivity of this detection platform was as low as 8.14 copies per μL, with a detection limit of 0.1%. These results demonstrated the superior performance of the platform, including high sensitivity, low detection limit, and wide applicability, enabling precise quantification and assessment of gene editing efficiency. In conclusion, microfluidic chip-based dPCR was used as a powerful tool for precise quantification and assessment of gene editing products.

The gene editing technology represented by clustered rule-interspersed short palindromic repeats (CRISPR)/Cas9 has developed as a common tool in the field of biotechnology. Many gene-edited products in plant varieties have recently been commercialized. However, the rapid on-site visual detection of gene-edited products without instrumentation remains challenging. This study aimed to develop a novel and efficient method, termed the CRISPR/SpRY detection platform, for the rapid screening of CRISPR/Cas9-induced mutants based on CRISPR/SpRY-mediated in vitro cleavage using rice (Oryza sativa L.) samples genetically edited at the TGW locus as an example. We designed the workflow of the CRISPR/SpRY detection platform and conducted a feasibility assessment. Subsequently, we optimized the reaction system of CRISPR/SpRY, and developed a one-pot CRISPR/SpRY assay by integrating recombinase polymerase amplification (RPA). The sensitivity of the method was further verified using recombinant plasmids. The proposed method successfully identified various types of mutations, including insertions, deletions (indels), and nucleotide substitutions, with excellent sensitivity. Finally, the applicability of this method was validated using different rice samples. The entire process was completed in less than an hour, with a limit of detection as low as 1%. Compared with previous methods, our approach is simple to operate, instrumentation-free, cost-effective, and time-efficient. The primary significance lies in the liberation of our developed system from the limitations imposed using protospacer adjacent motif sequences. This expands the scope and versatility of the CRISPR-based detection platform, making it a promising and groundbreaking platform for detecting mutations induced by gene editing.

Current research endeavors have focused on the combination of various isothermal nucleic acid amplification methods with CRISPR/Cas systems, aiming to establish a more sensitive and reliable molecular diagnostic approach. Nevertheless, most assays adopt a two-step procedure, complicating manual operations and heightening the risk of contamination. Efforts to amalgamate both assays into a single-step procedure have faced challenges due to their inherent incompatibility. Furthermore, the presence of the protospacer adjacent motif (PAM) motif (e.g., TTN or TTTN) in the target double-strand DNA (dsDNA) is an essential prerequisite for the activation of the Cas12-based method. This requirement imposes constraints on crRNA selection. To overcome such limitations, we have developed a novel PAM-free one-step asymmetric recombinase polymerase amplification (RPA) coupled with a CRISPR/Cas12b assay (OAR-CRISPR). This method innovatively merges asymmetric RPA, generating single-stranded DNA (ssDNA) amenable to CRISPR RNA binding without the limitations of the PAM site. Importantly, the single-strand cleavage by PAM-free crRNA does not interfere with the RPA amplification process, significantly reducing the overall detection times. The OAR-CRISPR assay demonstrates sensitivity comparable to that of qPCR but achieves results in a quarter of the time required by the latter method. Additionally, our OAR-CRISPR assay allows the naked-eye detection of as few as 60 copies/μL DNA within 8 min. This innovation marks the first integration of an asymmetric RPA into one-step CRISPR-based assays. These advancements not only support the progression of one-step CRISPR/Cas12-based detection but also open new avenues for the development of detection methods capable of targeting a wide range of DNA targets.

Introduction Genetically modified (GM) crops have been widely cultivated across the world and the development of rapid, ultrasensitive, visual multiplex detection platforms that are suitable for field deployment is critical for GM organism regulation. Objective In this study, we developed a novel one-pot system, termed MR-DCA (Multiplex RPA and Dual CRISPR assay), for the simultaneous detection of CaMV35S and NOS genetic targets in GM crops. This innovative approach combined Multiplex RPA (recombinase polymerase amplification) with the Dual CRISPR (clustered regularly interspaced short palindromic repeat) assay technique, to provide a streamlined and efficient method for GM crop detection. Methods The RPA reaction used for amplification CaMV35S and NOS targets was contained in the tube base, while the dual CRISPR enzymes were placed in the tube cap. Following centrifugation, the dual CRISPR (Cas13a/Cas12a) detection system was initiated. Fluorescence visualization was used to measure CaMV35S through the FAM channel and NOS through the HEX channel. When using lateral flow strips, CaMV35S was detected using rabbit anti-digoxin (blue line), whilst NOS was identified using anti-mouse FITC (red line). Line intensity was quantified using Image J and depicted graphically. Results Detection of the targets was completed in 35 min, with a limit of detection as low as 20 copies. In addition, two analysis systems were developed and they performed well in the MR-DCA assay. In an analysis of 24 blind samples from GM crops with a wide genomic range, MR-DCA gave consistent results with the quantitative PCR method, which indicated high accuracy, applicability and semi-quantitative ability. Conclusion The development of MR-DCA represents a significant advancement in the field of GM detection, offering a rapid, sensitive and portable method for multiple target detection that can be used in resource-limited environments.

The gene editing technology represented by clustered rule-interspersed short palindromic repeats (CRISPR)/Cas9 has developed as a common tool in the field of biotechnology. Many gene-edited products in plant varieties have recently been commercialized. However, the rapid on-site visual detection of gene-edited products without instrumentation remains challenging. This study aimed to develop a novel and efficient method, termed the CRISPR/SpRY detection platform, for the rapid screening of CRISPR/Cas9-induced mutants based on CRISPR/SpRY-mediated in vitro cleavage using rice (Oryza sativa L.) samples genetically edited at the TGW locus as an example. We designed the workflow of the CRISPR/SpRY detection platform and conducted a feasibility assessment. Subsequently, we optimized the reaction system of CRISPR/SpRY, and developed a one-pot CRISPR/SpRY assay by integrating recombinase polymerase amplification (RPA). The sensitivity of the method was further verified using recombinant plasmids. The proposed method successfully identified various types of mutations, including insertions, deletions (indels), and nucleotide substitutions, with excellent sensitivity. Finally, the applicability of this method was validated using different rice samples. The entire process was completed in less than an hour, with a limit of detection as low as 1%. Compared with previous methods, our approach is simple to operate, instrumentation-free, cost-effective, and time-efficient. The primary significance lies in the liberation of our developed system from the limitations imposed using protospacer adjacent motif sequences. This expands the scope and versatility of the CRISPR-based detection platform, making it a promising and groundbreaking platform for detecting mutations induced by gene editing.

Genetically modified (GM) foods have not only improved the yield and quality of food but also raised public concerns about the safety of GM foods. However, with the continuous innovation of sequencing technology, the characterization of GM organisms (GMOs) has been rapidly developed and upgraded several times. The traditional strategy of Southern blotting to confirm the copy number and Sanger combined with polymerase chain reaction (PCR) to identify the flanking sequences can accomplish the identification of goals, but it is not able to obtain the complete insertion information and is time-consuming and labor-intensive. Next-Generation DNA Sequencing (NGS) has effectively addressed this issue through its high-throughput sequencing technology, allowing for the rapid and cost-effective sequencing of large amounts of DNA. Its high-throughput sequencing technology has effectively addressed the limitations of traditional identification methods, offering a promising avenue for the characterization of GMOs. However, NGS is limited by the short read length and cannot identify and characterize all insertion sites included in the sequence data, especially in samples with relatively large and complex genomes. Third-generation DNA sequencing (TGS) breaks this limitation with the advantage of long reads, but its higher error rate and fewer bioinformatics tools are still the primary problems to be solved. This article presents a comprehensive review of the DNA-based techniques for assessing the molecular characterization of GM products since their introduction. The iterative evolution of these techniques is discussed, and the advantages and limitations of each method are analyzed. Through this analysis, the article identifies significant challenges and future development directions in the field of molecular characterization of GM products. By examining the strengths and weaknesses of current approaches, this review aims to provide insights and guidance for future research in this area.

CRISPR/Cas12a technology is used for nucleic acid detection due to its specific recognition function and non-specific single-stranded DNA cleavage activity. Here, we developed a fluorescence visualisation detection method based on PCR and CRISPR/Cas12a approaches. The method was used to detect the nopaline synthase terminator (T-nos) of genetically modified (GM) crops, circumventing the need for expensive instruments and technicians. For enhanced sensitivity and stability of PCR-CRISPR/Cas12a detection, we separately optimised the reaction systems for PCR amplification and CRISPR/Cas12a detection. Eleven samples of soybean samples were assessed to determine the applicability of the PCR-CRISPR/Cas12a method. The method could specifically detect target gene levels as low as 60 copies in the reaction within 50 min. In addition, accurate detection of all 11 samples confirmed the applicability. The method is not limited by large-scale instruments, making it suitable for mass detection of transgenic components in plants in the field. In conclusion, we developed a new, accurate, rapid, and cost-effective method for GM detection.

Effective regulation of gene-edited products and resolution of public concerns are the prerequisites for the industrialization of gene-edited crops and their derived foods. CRISPR-associated protein, the core element of the CRISPR system, requires to be regulated. Thus, there is an urgent need to establish qualitative and quantitative detection methods for the Cas gene. In the present study, the primers and probes were designed and screened for Cas12a (Cpf1), which is the most commonly used target site in gene editing; we performed PCR system optimization, determined the optimal primer concentration and annealing temperature, and established qualitative PCR and quantitative PCR (qPCR) assays for detecting Cpf1 in gene editing by specificity and sensitivity tests. In specificity testing, qualitative PCR and qPCR methods could 100% detect samples containing Cpf1 DNA, while the detection rate of other samples without Cpf1 was 0%. In the assay sensitivity test, the limit of detection of qualitative PCR was 0.1% (approximately 44 copies), and the limit of detection of the qPCR method was 14 copies. In the stability test, both the qualitative PCR and qPCR methods were repeated 60 times at their corresponding lowest detection limit concentrations, and the results were positive. Thus, the qualitative and quantitative assays for Cpf1 are specific, sensitive, and stable. The method provides technical support for the effective monitoring of gene-edited products and their derived foods in the future.

The safety and unintended effects of genetically modified (GM) crops have been the focus of public attention. Transcriptome analysis is a powerful tool to assess the potential impact of genetic modification on plant genomes. In this study, three transgenic (KMD, KF6, and TT51-1) and three non-transgenic (XS11, MH86, and MH63) rice varieties were assessed at the genomic and protein levels. The results of polymerase chain reaction (PCR) and Cry1Ab/1Ac speed test strips showed that the Bt gene was successfully expressed in transgenic rice. The results of RNA-seq analysis to analyze the unintended effects of transgenic Bt rice showed fewer differentially expressed genes (DEGs) between the transgenic and non-transgenic rice varieties than among the different varieties. Meanwhile, the results of principal component analysis and cluster analysis found no significant genetic variation between the transgenic and non-transgenic rice varieties, except for the presence of Bt in transgenic rice. There were only two co-upregulated DEGs and no co-downregulated DEGs among three comparison groups. Although there were various DEGs among the groups, the two co-upregulated DEGs were not related to any significantly enriched gene ontology (GO) term or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, indicating that the differences among the subgroups were more likely caused by complex environmental or genetic factors, rather than unintended effects due to Bt expression. This study provides useful information to further explore the unexpected effects and safety of GM rice.

Detecting short genetically modified (GM) nucleic acid fragments in GM crops and associated products is critically important for the global agriculture industry. Although nucleic acid amplification-based technologies have been widely used for genetically modified organism (GMO) detection, they still struggle to amplify and detect these ultra-short nucleic acid fragments in highly processed products. Here, we used a multiple-CRISPR-derived RNA (crRNA) strategy to detect ultra-short nucleic acid fragments. By combining confinement effects on local concentrations, an amplification-free CRISPR-based short nucleic acid (CRISPRsna) system was established to detect the cauliflower mosaic virus 35S promoter in GM samples. Moreover, we demonstrated assay sensitivity, specificity, and reliability by directly detecting nucleic acid samples from GM crops with a wide genomic range. The CRISPRsna assay avoided possible aerosol contamination from nucleic acid amplification and saved time due to an amplification-free approach. Given that our assay displayed distinct advantages over other technologies in detecting ultra-short nucleic acid fragments, it may have wide applications for detecting GM in highly processed products.

对基因编辑产品进行有效监管、消除公众疑虑是基因编辑产品在我国产业化的前提.目前,Cas9核酸内切酶是基因编辑技术中最常用的手段,因此需要建立针对Cas9核酸内切酶的定性定量检测方法.本研究以基因编辑中最常用的Cas9核酸内切酶外源序列为靶位点,设计和筛选引物、探针,进行PCR体系优化,确定最佳的引物浓度与退火温度,通过特异性、灵敏性等测试建立了检测基因编辑外源基因Cas9的普通PCR和qPCR检测方法.在检测方法特异性实验中,普通PCR和qPCR方法均只在含有Cas9等外源DNA的样品中扩增出了阳性结果,而其他不含有Cas9核酸内切酶的样品则均为阴性结果;在方法灵敏性测试中,普通PCR检测方法的检出限为0.1％,qPCR法检测下限(limit of detection,LOD)为16个拷贝;在方法稳定性实验中,普通PCR和qPCR方法在其相应最低检出限浓度下分别进行了67与60次重复实验,结果均为阳性.该定性定量方法适用于不同基因编辑作物,如水稻(Oryza sativa)、大豆(Glycine max)和油菜(Brassica napus)等.本研究建立的Cas9定性及定量检测方法特异性良好、灵敏度较高、稳定性较强.该方法的建立为将来实现对基因编辑产品的有效监测提供了一定的技术支撑.

The insertion position of the exogenous fragment sequence in a genetically modified organism (GMO) is important for the safety assessment and labeling of GMOs. SK12-5 is a newly developed transgenic maize line transformed with two trait genes [i.e., G10evo -5-enolpyrul-shikimate-3-phosphate synthase (EPSPS) and Cry1Ab/Cry2Aj ] that was recently approved for commercial use in China. In this study, we tried to determine the insertion position of the exogenous fragment for SK12-5. The transgene–host left border and right border integration junctions were obtained from SK12-5 genomic DNA by using the thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and next-generation Illumina sequencing technology. However, a Basic Local Alignment Search Tool (BLAST) analysis revealed that the flanking sequences in the maize genome are unspecific and that the insertion position is located in a repetitive sequence area in the maize genome. To locate the fine-scale insertion position in SK12-5, we combined the methods of genetic mapping and nanopore-based sequencing technology. From a classical bulked-segregant analysis (BSA), the insertion position in SK12-5 was mapped onto Bin9.03 of chromosome 9 between the simple sequence repeat (SSR) markers umc2337 and umc1743 (26,822,048–100,724,531 bp). The nanopore sequencing results uncovered 10 reads for which one end was mapped onto the vector and the other end was mapped onto the maize genome. These observations indicated that the exogenous T-DNA fragments were putatively integrated at the position from 82,329,568 to 82,379,296 bp of chromosome 9 in the transgenic maize SK12-5. This study is helpful for the safety assessment of the novel transgenic maize SK12-5 and shows that the combined method of genetic mapping and the nanopore-based sequencing technology will be a useful approach for identifying the insertion positions of transgenic sequences in other GM plants with relatively large and complex genomes.

肉类掺假问题直接影响着人类健康、公共卫生安全以及社会稳定等方面,成为当今食品安全热点话题之一,因此,高效、精确的肉类及肉制品中动物源性成分的检测鉴定势在必行.基于此,主要介绍了对于动物源性成分检测及鉴别的不同研究方法,分析了利弊,并对后续肉类及肉制品中动物源性成分的鉴别方法的研发方向进行了展望,以期为此领域提供资料性参考.

Gene-editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been reported that detect mutations at targeted loci induced by the CRISPR/Cas system in different organisms, they are semiquantitative and have difficulty in the detection of mutants in processed food samples containing low initial concentrations of DNA and may not accurately quantify editing frequency, especially at very low frequencies in a complex polyploid plant genome. In this study, we developed a duplexed dPCR-based method for the detection and evaluation of gene-editing frequencies in plants. We described the design, performance, accurate quantification, and comparison with other detection systems. The results show that the dPCR-based method is sensitive to different kinds of gene-editing mutations induced by gene-editing. Moreover, the method is applicable to polyploid plants and processed food samples containing low initial concentrations of DNA. Compared with qPCR and NGS-based methods, the dPCR method has a lower limit of detection (LOD) of the editing frequency and a better relationship with the expected editing frequency in detecting the edited region of gene-edited rice samples. Taken together, the duplexed dPCR assay is accurate and precise, and it will be a powerful tool for the detection and evaluation of gene-editing frequencies in plants in gene-editing technology.

Genetically modified organisms (GMOs) need to be evaluated for safety before their release. Metabolome techniques provide effective methods to evaluate unintended effects. In this study, the grain metabolome of six transgenic rice lines containing secticidal cry and glyphosate tolerance epsps genes were tested by using an non-targeted metabolite profiling, and 161 and 138 metabolites were identified in grains at grain-filling stage and mature stage, respectively. The metabolic profiles of genetically modified (GM) lines and non-GM lines were all significantly different from each other at grain-filling stage. Although the levels of many metabolites were significantly changed in each transgenic line, only seven of them were simultaneously changed in all the six lines compared with those in non-GM lines at grain-filling stage. The number of significantly changed metabolites was much less at mature stage than that at grain-filling stage. Besides, none of these metabolites was simultaneously changed in all the six lines, suggesting that the metabolites of GMOs are different at different stages of GMO development. This study provides useful information about the metabolic variation between GMO and non-GMO in two development stages of rice grains. These findings suggest that non-targeted metabolite profiling can strengthen the assessment of risk based on metabolites.

基因编辑技术指能够对目标基因进行编辑,实现对特定DNA片段的敲除、 加入等技术.目前,该技术主要包括锌指蛋白系统、 转录激活因子样效应核酸酶系统和成簇的规律间隔短回文重复序列系统,基本原理都是通过序列特异性核酸酶特异切割DNA靶位点,产生DNA双链断裂,诱导DNA的损伤修复机制,从而实现对指定基因组的定向编辑.本文概述了3种基因编辑技术的原理,对它们的优缺点进行比较,并阐述了CRISPR基因编辑技术在动物和植物育种中的应用,以及基于CRISPR技术的DNA分子检测新方法的建立和应用.其次,阐述了对CRISPR等基因编辑产品筛选和检测鉴定的方法,比较了几种筛选方法的检测周期、 灵敏性、 人工成本等.最后,对基因编辑技术的发展前景进行了展望.