Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR/Cas12a detection platform.

CRISPR/Cas12a is a highly promising detection tool. However, detecting single nucleotide variations (SNVs) remains challenging. Here, we elucidate Cas12a specificity through crRNA engineering and profiling of single- and double-base mismatch tolerance across three targets. Our findings indicate that Cas12a specificity depends on the number, type, location, and distance of mismatches within the R-loop. We also find that introducing a wobble base pair at position 14 of the R-loop does not affect the free energy change when the spacer length is truncated to 17 bp. Therefore, we develop a new universal specificity enhancement strategy via iterative crRNA design, involving truncated spacers and a wobble base pair at position 14 of the R-loop, which tremendously increases specificity without sacrificing sensitivity. Additionally, we construct a PAM-free one-pot detection platform for SARS-CoV-2 variants, which effectively distinguishes SNV targets across various GC contents. In summary, our work reveals new insights into the specificity mechanism of Cas12a and demonstrates significant potential for in vitro diagnostics.