2.1 crRNA screening
Four groups of crRNAs (IS711-crRNA-1-4) (Table 2) were compared by the method of RPA-Cas12a, and show four groups of crRNA binding sites are indicated below (Figure 2A). The Cas12a cleavage products will be checked by electrophoresis on 2% agarose gel to exhibit the cleavage efficiency (Figure 2B). Moreover, Figure 2C exhibited the endpoint fluorescence value with a real-time PCR instrument. The highest efficient crRNA was selected by the above two approaches. The results are presented below (Figure 2B). The gel electrophoresis of IS711-crRNA-1,2 and 3 Cas digestion products showed complete cleavage compared to IS711-crRNA-4. The fluorescence quantification showed the highest fluorescence value at the IS711crRNA-2 endpoint. Therefore, all subsequent experiments were performed by adopting IS711-crRNA-2 (uaauuucuacuaaguguagauGAUGAAUCCGUCACGCUCGG).