2.3. The design of the CRISPR/Cas12a system
The Cas12a protein functions as molecular ”scissors,” precisely cleaving ssDNA sequences. These DNA-targeting crRNAs bind with Cas12a, forming a complex known as Cas12a-crRNA. This complex acts as intelligent scissors, capable of cleaving the intended DNA target while sparing non-targets, even when there’s a single base mismatch. The precision of the CRISPR/Cas12a system heavily relies on meticulous design and selection of crRNA for specific applications. To evaluate the system’s specificity, we investigated the binding of crRNAs with mismatched bases at various positions to the original SARS-CoV-2 cDNA templates, with a concentration of 1 nM (as shown in Figure 5A). Notably, introducing a mismatch at either the 1st or 3rd base of the protospacer (crRNA-mis1 and crRNA-mis3) significantly diminished fluorescence signals, though not to a negligible level. As the mismatch was positioned further from the initiation site of the protospacer, the reduction in fluorescence became less pronounced. The impact of crRNA mismatches at positions 11 to 15 resembled that of the original crRNA. Remarkably, a mismatch at the 3rd site of the protospacer yielded the most minimal trans-cleavage activity. Based on these findings, we propose that the SNP site, such as the L452R mutation, could be strategically positioned at the 3rd site of the protospacer within the crRNA sequence. This arrangement would maximize the differentiation between on-target and off-target samples. However, it’s crucial to note that the off-target trans-cleavage activity does affect the accuracy of detecting the L452R mutation.
To address this challenge, a novel approach termed ”dual mismatch” was implemented in the subsequent experiment (Figure 5B). For the BA.5 cDNA template, crRNA-mis3 was identified as the appropriate crRNA sequence. The BA.2 cDNA template was considered analogous to the ”wild-type” sequence due to the absence of mutation sites within this range. An additional mismatch was introduced in the 1st to 9th sites of the protospacer, resulting in a series of crRNAs labeled as ”crRNA-mis3 + 1” to ”crRNA-mis3 + 9”. Each of these modified crRNAs was tested against both the BA.2 and BA.5 cDNA templates. When mismatches were introduced at positions 1 to 5 of the protospacer, almost no detectable fluorescence signal was observed during the assay with the BA.2 template. However, a mismatch at the 5th site still resulted in a relatively strong fluorescence signal when tested against the BA.5 template (approximately 52.27% of the signal from a perfect match). Based on these outcomes, the crRNA-mis3 + 5 emerged as the optimal crRNA for targeting BA.5 cDNA sequences with the capability to discern single-nucleotide changes. Following the successful design of the CRISPR/Cas12a system for enhanced cDNA sequence specificity, a robust photoelectrochemical platform was established to further achieve high sensitivity in subsequent steps.