Artemisinin resistance in Plasmodium falciparum is closely linked to mutations in the kelch13 gene, particularly dual-base substitutions at codon 613 (Q613E/Q613H). Traditional PCR and sequencing methods cannot reliably distinguish these mutations at the same site, while existing CRISPR-Cas12a–based multiplex assays often rely on additional equipment such as quantum dots or microfluidic chips, making them difficult to deploy in resource-limited settings.

A research team led by Chinese scientists, recently published a paper in Microchemical Journal entitled “Mismatch crRNA mediated CRISPR/Cas12a system to simultaneously screen dual adjacent mutations of kelch13 gene in Plasmodium falciparum resistance to artemisinin.”

In this work, the team developed a novel “mismatch crRNA” strategy that enables one-pot discrimination of Q613 wild-type, Q613E, and Q613H variants. This approach allows rapid, low-cost, and highly sensitive genotyping of single amino acid substitutions at codon 613 of kelch13, providing a powerful tool for monitoring artemisinin resistance in malaria-endemic regions such as Africa, and offering a paradigm for detecting complex mutations in other pathogens.

EDITGENE is proud to have supported this research by providing high-performance Cas12a proteins, contributing to the successful outcome of the study.

Spotlight

The study introduces a mismatch crRNA strategy that enables the CRISPR/Cas12a system to distinguish three kelch13 genotypes—Q613, Q613E, and Q613H—within a single-tube reaction.

2. Detection advantages: sensitive, rapid, and cost-effective

The assay delivers results in just 30 minutes, detects mutations at concentrations as low as 20 pM, and accurately differentiates dual mutations with outcomes consistent with Sanger sequencing, making it practical for use in resource-limited settings.

3. Application potential: malaria resistance surveillance and control

Validated in 28 clinical samples, the method provides an effective tool for monitoring artemisinin resistance–associated mutations and offers scalability to other drug-resistance loci.

Experimental validation confirmed that mismatch-designed crRNAs can specifically distinguish between wild-type and mutant kelch13 Q613 variants (Q613E/Q613H). For the wild-type Q613, the presence of double mismatches failed to activate Cas12a, yielding signals comparable to the negative control. In contrast, Q613E and Q613H produced strong fluorescence signals, with Q613H generating a higher signal intensity due to its mismatch position being farther from the PAM site.

For low-abundance mutations, PCR pre-amplification was required for effective discrimination. The results demonstrated high specificity and improved detection sensitivity, enabling reliable identification of mutations with frequencies as low as 0.1%.

In summary, the mismatch crRNA-mediated CRISPR/Cas12a system allows highly specific detection of Q613 mutations, though trace-level detection remains dependent on pre-amplification.

Through systematic optimization, the researchers established a high-performance CRISPR/Cas12a detection system. Key parameters include:

Together, these optimized conditions significantly enhanced the overall performance and reliability of the detection system.

The mismatch crRNA-mediated CRISPR/Cas12a system was applied to genotype the kelch13 gene in 28 clinical samples:

These findings demonstrate the excellent accuracy and reliability of the proposed method in real clinical samples, underscoring its potential as a robust tool for on-site surveillance of artemisinin resistance in malaria-endemic regions.