A CRISPR/Cas12a-Assisted in Vitro Diagnostic Tool for Identification and Quantification of Single CpG Methylation Sites

Cancer, as a disease, starts with a change in the genome that results in the downregulation of genes involved in "normal" cell behavior. This genetic change can either be induced by a change in the DNA code (mutation) or a change that does not affect the genetic code but instead influences the way this code can be read. The latter is called an epigenetic alteration. While cytosine methylation is a healthy process, methylation of promoter CpG islands has also been shown to be a critical hallmark in many cancer cells. This is because specific genes on the DNA must always be expressed to prevent a "normal" cell from becoming a cancer cell. With the upcoming field of liquid biopsies and the conformation that these hypermethylated DNA sequences could be found in urine, the development of novel biosensors for fast and reliable detection of these sequences becomes of interest. Integrating CRISPR/Cas technology in miniaturized sensors for on-site analysis is very promising for biomarker detection at the point of care. However, while CRISPR sensing seems a promising alternative to established DNA detection techniques, it is not sensitive to epigenetic changes like hypermethylation. In this presentation, we showcase a method to make Cas12a sensitive to CpG methylation, which is based on the addition of methylation-sensitive restriction enzymes to the CRISPR sensing process. Furthermore, we discuss the effect of DNA fragmentation on the cleavage activity of Cas12a and how this can be used in biosensing.

Learning Objectives:

1. Discuss the mechanism of Cas12a and how this can be used for biosensing of dsDNA sequences.

2. Identifying the difference in Cas12a activity for different target sequence lengths.  

3. Infer that, by selecting the right enzymes and crRNA sequences, Cas12a combined with methylation-sensitive restriction enzymes can be used to determine the methylation state of a single CpG methylation site.