Advances in the application of recombinase-aided amplification combined with CRISPR-Cas technology in quick detection of pathogenic microbes

Abstract

The rapid diagnosis of pathogenic infections plays a vital role in disease prevention, control, and public health safety. Recombinase-aided amplification (RAA) is an innovative isothermal nucleic acid amplification technology capable of fast DNA or RNA amplification at low temperatures. RAA offers advantages such as simplicity, speed, precision, energy efficiency, and convenient operation. This technology relies on four essential components: recombinase, single-stranded DNA-binding protein (SSB), DNA polymerase, and deoxyribonucleoside triphosphates, which collectively replace the laborious thermal cycling process of traditional polymerase chain reaction (PCR). In recent years, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated proteins) system, a groundbreaking genome engineering tool, has garnered widespread attention across biotechnology, agriculture, and medicine. Increasingly, researchers have integrated the recombinase polymerase amplification system (or RAA system) with CRISPR technology, enabling more convenient and intuitive determination of detection results. This integration has significantly expanded the application of RAA in pathogen detection. The step-by-step operation of these two systems has been successfully employed for molecular diagnosis of pathogenic microbes, while the single-tube one-step method holds promise for efficient pathogen detection. This paper provides a comprehensive review of RAA combined with CRISPR-Cas and its applications in pathogen detection, aiming to serve as a valuable reference for further research in related fields.

Keywords: clustered regularly interspaced short palindromic repeats associated proteins; pathogenic microbes; quick detection; recombinase-aided amplification; specific diagnosis.

FIGURE 1

Schematic diagram of RAA implementation for real-time detection. This figure was drawn by Figdraw. The single fluorogenic probe includes fluorophor, quencher and 3′block, and exonuclease cannot recognize the single fluorogenic probe. As the probe binds to the target DNA, exonuclease recognizes tetrahydrofuran on the probe abasic site and cleavage them, allowing the fluorophor and quencher to separate and fluoresce. At the same time, DNA polymerase unblocks the 3′block and completes the chain extension.

FIGURE 2

Schematic diagram of single-tube one-step method. This figure was drawn by Figdraw. The specific principle of single-tube one-step detection of RNA viruses is as follows: in the nucleic acid amplification system under constant temperature conditions, firstly, dNTP is used as substrate material, mRNA is used as a template, and under the action of reverse transcriptase, a cDNA single-strand complementary to the RNA template is synthesized to form DNA-RNA heterozygote. In this DNA-RNA heterozygote, RNA is specifically degraded by reverse transcriptase, and then dNTP is used as the substrate, the first strand of cDNA is used as a template, and under the action of DNA polymerase, the second strand of cDNA is synthesized, and finally the double-stranded DNA molecule is formed, that is, the DNA synthesis process guided by RNA is completed. Then, this double-stranded DNA molecule is used as the template DNA. When the primer finds the complementary sequence that perfectly matches the template DNA, with the help of recombinase, the double-stranded structure of the template DNA is opened, while SSB stabilizes the displaced DNA chain, and the extension of the strand is completed under the action of DNA polymerase to form a new complementary strand of DNA. The T7 RNA polymerase transcribed the amplified product into RNA, while the Cas13a protein specifically bound the target nucleic acid under the guidance of crRNA, the protein structure was changed, and was converted into ribonuclease, which was non-specific to cut RNA. The incidental cutting activity could cut the introduced externally derived FQ-coated ssRNA probe. The fluorescence signal can be amplified rapidly to detect the target nucleic acid quickly.