Mismatch-Enhanced Specific PCR (MES-PCR): A Rapid and Cost-Effective Method for Screening CRISPR/Cas9-Induced Mutations

One-pot SP-EXPAR-integrated CRISPR/Cas14a assay for highly specific and versatile detection of ctDNA mutations in lung cancer patients.

The highly specific and versatile detection of KRAS mutations in circulating tumor DNA (ctDNA) from plasma has critical clinical implications for non-small-cell-lung cancer (NSCLC). However, conventional isothermal amplification methods suffer from poor single-base discrimination, while CRISPR-12a-based detection is highly protospacer adjacent motif (PAM)-dependent. To address these challenges, a one-pot self-primer isothermal exponential amplification reaction (SP-EXPAR) combined with a CRISPR/Cas14a assay was developed for detecting KRAS G12C and G12D. Two synergistic strategies were devised to ensure high specificity: first, a carefully designed hairpin probe that permits selective amplification of mutant over wild-type sequences through differential binding affinity; second, optimization of the Cas14a sgRNA seed region, with the mutation positioned at the 11th nucleotide for stringent target recognition. The assay is further distinguished by a physical separation design, in which the Cas14a reagents are pre-loaded into the tube cap and mixed with the amplification products only after SP-EXPAR completion. This assay enables KRAS G12C detection within 1 h, with a limit of detection of 81.9 aM (0.1% mutation percentage) and a dynamic range from 100 aM to 1 nM. Furthermore, this assay further demonstrates its programmability and was successfully applied to detect KRAS G12D with comparable performance. In detecting 42 clinical samples, this assay demonstrated 100% sensitivity and 100% specificity compared with DNA sequencing. This approach holds great potential in disease diagnosis.

Rapid PCR-based screening system for detection of type II CRISPR-Cas loci in bacterial species

CRISPR/Cas9-Mediated Site-Directed Mutagenesis of Genes in Klebsiella pneumoniae.

This protocol minimizes off-target editing, shortens experimental timelines, reduces screening workload, and provides a reliable platform for investigating resistance mechanisms, validating candidate genes, and supporting functional genomics studies in clinically relevant bacterial pathogens.

Easy detection of CRISPR/Cas9-Induced Insertions, Deletions, and Substitutions in Rice Genes OsMADS26, OsRAC1 and OsNRT1.1b using SYBR Green qPCR and Robust HRM analysis in R software.

Rice is a major cereal crop for global food and nutritional security and a key target for genetic improvement. CRISPR/Cas9 enables precise genetic modification in crops, but mutation screening remains a technical and economic barrier to broader genome-editing applications. Although several detection methods are available, some require labor-intensive procedures, specialized equipment, high costs, or limited sensitivity to specific mutation types. High-resolution melting (HRM) analysis is an established approach for screening CRISPR/Cas-induced mutations in plants, including rice. Here, we evaluated an adapted HRM workflow combining conventional SYBR Green-based qPCR chemistry with downstream computational analysis to detect CRISPR/Cas9-induced mutations at three rice loci: OsMADS26, OsRAC1, and OsNRT1.1b. The workflow detected insertions, deletions, and base substitutions. Across the three loci, the 1% edited-DNA mixtures showed a slight observable deviation from the wild-type melting profile under the conditions evaluated, although this should not be interpreted as a validated detection threshold. Although the assessment of heterozygous samples was limited by their availability, the results support the potential applicability of the approach for individual sample analysis and expanded sample screening. A customizable R script complemented visual analysis by evaluating melting temperature (Tm) and GCP-derived dissimilarity, supporting sample classification. By combining standard SYBR Green chemistry with an adaptable analysis workflow, the method offers an alternative to dedicated HRM reagents and proprietary platforms. This approach provides a practical and potentially lower-cost option for mutation screening at the evaluated rice loci and may be adapted and validated for other targets and plant species.

Integration of PNA-mediated PCR and CRISPR/Cas13a for highly sensitive detection of EGFR T790M mutation in circulating tumor DNA.

Circulating tumor DNA (ctDNA) is characterized by low abundance and fragmentation, limiting the development of genetic variant detection technologies. In this study, we established a highly sensitive and specific assay by combining peptide nucleic acid (PNA)-mediated PCR clamping with CRISPR/Cas13a trans-cleavage detection. A PNA probe targeting the wild-type (WT) EGFR T790M allele was designed to suppress WT amplification during PCR, while minimally affecting mutant allele amplification. By combining the target specificity of Cas13a for mutant alleles with the WT-suppression capability of PNA-PCR, we achieved a dual-enrichment effect for mutant detection. When applied to EGFR T790M mutation detection, the assay reached a analytical sensitivity of 0.02%. We established a standard curve for T790M detection using cell-free DNA standards. Clinical validation in 20 plasma samples from lung adenocarcinoma patients demonstrated that the PNA-Cas13a assay achieved a diagnostic sensitivity of 93.3% (95% CI: 68.1%-99.8%) and a specificity of 100% (95% CI: 47.8%-100%), with detection concordance comparable to or improved over ARMS-PCR in this pilot cohort. The result suggest its preliminary diagnostic utility in liquid biopsy. In conclusion, the PNA-Cas13a assay enables sensitive and specific detection of EGFR T790M mutations in ctDNA, is readily adaptable to multiple gene loci, and holds promise for clinical monitoring of tumor drug resistance.

TOPS-CRISPR: Thermally-regulated and oligonucleotide-mediated one-pot CRISPR-Cas12a assay for ultra-sensitive and rapid on-site diagnostics.

CRISPR-Cas12a has emerged as a powerful tool in molecular diagnostics, owing to its robust signal amplification and compact crRNA design. However, its uncontrolled enzymatic activity often hampers application in streamlined one-pot assays. Although existing temporal or spatial regulation strategies can mitigate this issue, they typically introduce operational complexity or increased cost. Here, we designed a Thermally regulated, Oligonucleotide-mediated one-Pot System for CRISPR-Cas12a (TOPS-CRISPR), which employs a programmable inhibition strategy based on complementary RNA blockers with tunable length and binding sites, enabling efficient and reversible steric inhibition of the LbCas12a-crRNA ribonucleoprotein (RNP) complex, resolving the inherent contradiction between amplification and cleavage in one-pot assay. TOPS-CRISPR not only is operational simple and cost-effective but also achieves over 60-fold higher sensitivity than conventional one-pot platforms. We demonstrated the clinical applicability of TOPS-CRISPR by accurately detecting Brucella and Streptococcus in both spiked and clinical samples. Moreover, the system integrates seamlessly with rapid sample processing, lyophilized reagents, and miniaturized workflows, enabling field-deployable pathogen identification within 50 min.