Protocol for high-precision CRISPR-Cas12a-based SNV detection on synthetic DNA, cell line cfDNA models, and liquid biopsies

Figures

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  Figure 1. Harvesting conditioned medium for cfDNA isolation During apoptosis, genomic DNA is fragmented and subsequently released into the surrounding medium. After approximately one week of incubation, the conditioned medium can be collected by centrifugation and filtration, ensuring the removal of any viable cells with intact genomic DNA from the final harvested medium.

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  Figure 2. Isolation of cfDNA from blood Non-coagulated blood is centrifuged to harvest the resulting plasma supernatant, containing cfDNA. cfDNA can be isolated from plasma using affinity-based purification protocols, such as QIAsymphony DSP DNA mini as described in this protocol.

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  Figure 3. Overview of crRNA design for LbCas12a-based detection (A) Cas12a interrogates dsDNA using the spacer sequence of the crRNA that hybridizes with the protospacer sequence on the DNA target strand [TS]. The first 5′ pentanucleotide stretch of the crRNA spacer forms the seed region. The shown direct repeat sequence is compatible with LbCas12a. The displayed spacer sequence corresponds to BRAF p.V600E, without synthetic mismatches. (B) Schematic overview of designed synthetic DNA oligonucleotide targets that contain ends with flanking genomic sequences.

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  Figure 4. Schematic overview of CRISPRdx testing on synthetic dsDNA oligonucleotides Duplex DNA formation of the ordered synthetic ssDNA nucleotides yields dsDNA targets that can be diluted to desired concentrations. CRISPRdx mastermix, consisting of Cas12a, crRNA and reporter in a buffered solution is distributed in a 384-well plate, followed by addition of target. Different combinations of crRNA and target will have to be tested to evaluate specificity improvement after engineering a crRNA with a synthetic mismatch [SM]. Reactions are monitored for fluorescence over time in a pre-heated plate reader.

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  Figure 5. Transfer of raw data from excel to GraphPad Prism software Raw fluorescence data is pasted into a XY table format, with replicates represented as adjacent columns. Time units have been adjusted accordingly.