Liquid biopsy continues to be a focus of the cancer diagnostics industry, primarily due to the non-invasive nature of sample collection, however the low levels of circulating tumor DNA (ctDNA) remain a challenge. Methylation has emerged as a promising biomarker for detecting ctDNA, yet detecting the cancerous signal in an excess of non-cancerous, unmethylated cfDNA in the background has been proven difficult. Harbinger Health has developed a novel method that depletes unmethylated background at the CpG-level from specific genomic regions of interest, resulting in an enrichment of the ctDNA fraction. We developed a background cfDNA depletion method that utilizes the endonuclease activity and the specificity of CRISPR/Cas12a to remove unmethylated DNA fragments at ctDNA methylation biomarker sites. First, we identified cancer-informative regions that showed consistent contiguous elevated methylation states in cancer compared to normal samples. We then designed guide RNAs that specifically bind and cut unmethylated CpG sites within the target regions. Cas depletion of bisulfite-converted DNA enriches cancer informative signal for bioanalysis. To test validity of our Cas complex designs, we created model samples that mimic fully unmethylated and fully methylated species. We spiked these cfDNA models into a background of genomic DNA from 5% to 0.01%. In triplicate, we examined cutting efficiency, multiplexing and specificity of Cas to unmethylated species of model DNA. Results were assessed using qPCR by comparing differences in Ct with and without Cas treatment. Additionally, we applied this depletion step during our library preparation process to show utility in methylation analysis of low tumor content liquid biopsy samples. Using both the model sample dilution series and bisulfite converted cfDNA samples, which represent a range of cancerous and background methylation signal, we deplete unmethylated targets using Cas prior to indexing PCR. This rendered unmethylated targets non-amplifiable, removing them from analysis. Results were assessed using both qPCR and sequencing data, such as methylated read counts over the target region. We demonstrated our analytical approach efficiently depletes non-cancer cfDNA by on average 9-fold, which increases the fraction of ctDNA signal and can be multiplexed. This background signal depletion driven by CRISPR is sensitive and specific, maintaining methylated DNA signal detection down to 0.01% at inputs of 10ng DNA. Our Cas-mediated depletion of background signal method has proven useful for enriching rare methylated fragments over background by improving the signal to noise ratio. This technique is novel by harnessing the specificity of CRISPR to target DNA in both a sequence and methylation state specific manner. The depletion of precise methylation states at the CpG-level make it suitable for use in multiple bioassays.

Citation Format: Caitlin Gilley, Miguel Williams, Emily Neaga, Sarah Falotico, Kade Pettie, Anthony Shuber. A methylation state specific targeted background depletion technique for enrichment of ctDNA fraction [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A014.