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.