Recently, several groups have shown the potential of the CRISPR/Cas12a system for sensitive and selective DNA detection. However, its application on single nucleotide variants (SNVs) detection is limited by the requirement of a protospacer adjacent motif (PAM) directly upstream to the SNV site and the amplification of non-specific signals due to the rapid and indiscriminate trans cleavage activity. As a useful application, the sensitive and accurate detection of rare mutations has profound clinical implications. However, current methods require expensive instrumentation, and they are laborious and time-consuming. Thus, there is a need for a probe-based alternative that can effectively discriminate single-base mutations with high sensitivity.

This thesis showcases a detection of rare mut...[ Read more ]

Recently, several groups have shown the potential of the CRISPR/Cas12a system for sensitive and selective DNA detection. However, its application on single nucleotide variants (SNVs) detection is limited by the requirement of a protospacer adjacent motif (PAM) directly upstream to the SNV site and the amplification of non-specific signals due to the rapid and indiscriminate trans cleavage activity. As a useful application, the sensitive and accurate detection of rare mutations has profound clinical implications. However, current methods require expensive instrumentation, and they are laborious and time-consuming. Thus, there is a need for a probe-based alternative that can effectively discriminate single-base mutations with high sensitivity.

This thesis showcases a detection of rare mutations and reports an ultra-selective Cas12a-based system that eliminates the need for the PAM sequence in the target with lower noise from the wild-type sequence by using its non-canonical double-stranded trans cleavage activity in chapter 2. A biosensor was developed using this strategy to allow the detection of an EGFR gene mutation in sub-femtomolar concentrations up to 0.1% variant allele frequency using optimized immobilization-free electrochemical based readout and fluorescence readout as a reference in chapter 3.

To develop a detection method with high sensitivity of plasma biopsy for cancer-related genotyping, in chapter 2 the ultra-sensitive and specific trans cleavage activity of AsCas12a toward double-stranded DNA was explored to be able to do detection of the 0.1% VAF EGFR T790M target gene in mock samples. As shown in chapter 3, in the absence of an amplification step of target DNA, the detection of the target gene can reach sub femtomolar. After asymmetric PCR amplification, the detection of the target gene can reach attomolar level, which shows a great clinical value for plasma biopsy of specific genotypes of cancer. However, due to the trans cleavage characteristics of AsCas12a protein, this study may have limitation in screening-based study which requires specific multi-signal readouts.

As future work, utilizing an immobilization-based electrochemical detection method explained in chapter 4, a biosensor with a multi-signal readout for POCT will be developed.