Integration of CRISPR/Cas12a and Multiplexed RPA for Fast Detection of Gene Doping

Abstract

Fast and on-site detection is important for an effective antigene-doping strategy. However, the current gene doping (GD) evaluation methods require sophisticated instruments and laborious procedures, limiting their field applications. This study proposes a CRISPR/Cas12a-based detection platform (termed CasGDP) combining CRISPR/Cas12a and multiplexed Recombinase Polymerase Amplification (RPA) for rapid evaluation of GD. CasGDP showed high specificity for identifying the putative target genes such as EPO, IGF-1, and GH-1. By using fluorescence as the readout, the method achieved a limit-of-detection of 0.1 nM and 1 aM for unamplified and amplified target plasmids, respectively. Additionally, an in vitro GD cell model was successfully established with the human EPO gene (hEPO). The results indicated that the hEPO gene transfection promoted the hEPO protein expression. Furthermore, trace amounts of EPO transgene spiked in human serum were efficiently measured by CasGDP with fluorescence-and lateral flow device (LFD)-based readouts in 40 min. Finally, we designed a multiplexed microfluidic device and realized simultaneous detection of the three transgenes via LFD embedded in the device. To our knowledge, this is the first work that combines the CRISPR-based system and multiplexed RPA for GD detection. We anticipate CasGDP to be widely used as a rapid, sensitive, and robust tool for GD evaluation.

Development of a Naked Eye CRISPR-Cas12a and -Cas13a Multiplex Point-of-Care Detection of Genetically Modified Swine

“…Compared to earlier CRISPR nucleic acid assays, this multiplex CRISPR nucleic acid assay mediated by the CRISPR-Cas12a and -Cas13a systems can simultaneously detect two target genes in a single tube reaction, reducing the cost of amplification and the number of tubes required, effectively reducing amplicon nucleic acid contamination from the environment. − The outstanding advantage of this multiplex CRISPR nucleic acid assay compared to other CRISPR colorimetric/fluorescence assays is the use of modified green-red-yellow, fluorescent signal conversion ssDNA-FQ and ssRNA-FQ reporters combinations suitable for visualization with the naked eye, eliminating the need for expensive microplate readers and specialized hand-held devices for visualization. − …”

“…The CRISPR-Cas12a and -Cas13a-based dual-channel system combined with multiplex RPA or recombinase-aided amplification (RAA) can rapidly detect nucleic acids. However, this approach relies on costly dual-channel fluorescence unsuitable for large-scale field applications. , We previously developed ssDNA-FQ reporters with JOE and ROX fluorophores to increase the fluorescent signal for easy visualization and differentiation during nucleic acid detection . Moreover, we evaluated the performance of 16 types of ssDNA-FQ reporters for use with CRISPR/Cas12a-based visual colorimetric and fluorescent assays .…”

Rapid and Sensitive Detection of Fungicide-Resistant Crop Fungal Pathogens Using an Isothermal Amplification Refractory Mutation System

“…Currently available techniques to detect crop fungal pathogens are mainly the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and isothermal nucleic acid amplification. − ELISA detects pathogens by identifying specific proteins rather than nucleic acid molecules, , so it is not suitable for identifying fungicide-resistant pathogens with only single-nucleotide mutation. Among all the molecular assays, PCR is the most widely used method due to its advantages in sensitivity, precision, and reagent availability. − Particularly, the emergence of isothermal amplification, such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), eliminates the need for costly temperature-control instruments, thus facilitating in-field testing. − However, neither of these nucleic acid amplification methods can directly detect genetic mutations. The sequencing technique allows for obtaining genetic information with a single-nucleotide resolution.…”

Unlocking the Full Potential of Cas12a: Exploring the Effects of Substrate and Reaction Conditions on Trans-Cleavage Activity

“…Normally, CRISPR-Cas12a is used in conjunction with pre-amplification methods such as polymerase chain reaction (PCR), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), and ligase chain reaction (LCR) . However, it has been reported that Cas12a exhibits low trans-cleavage activity by itself, and the detection limit for nucleic acid without pre-amplification only reached the nM level. , The relatively poor sensitivity of CRISPR-Cas12a has limited its further applications as pre-amplification methods require longer reaction times and more complex designs. , …”

“…Subsequently, numerous analytical methods were developed based on Cas12a's programmability and reactivity, 2−4 covering the detection of nucleic acids, 5,6 proteins, 7 small molecules, 7−9 ions, 10 and facilitating molecular diagnostics of COVID-19, 11−15 cancers, 16,17 and pathogens. 18,19 Normally, CRISPR-Cas12a is used in conjunction with preamplification methods such as polymerase chain reaction (PCR), 20 recombinase polymerase amplification (RPA), 21 loop-mediated isothermal amplification (LAMP), 22 rolling circle amplification (RCA), 23 and ligase chain reaction (LCR). 24 However, it has been reported that Cas12a exhibits low trans-cleavage activity by itself, and the detection limit for nucleic acid without pre-amplification only reached the nM level.…”