The CRISPR-Cas12a system is a new type of genome editing tool with high efficiency and targeting. However, other sequences in the genome may also be cleaved nonspecifically, resulting in unavoidable off-target effects. Therefore, it is necessary to learn more about the mechanism of CRISPR-Cas12a to recognize target sequences to avoid its off-target effects. Here, we show that insertion (DNA bubble) or deletion (RNA bubble) of the target dsDNA sequence compared with the crRNA sequence, the CRISPR-Cas12a system can still recognize and cleave the target dsDNA sequence. We conclude that the tolerance of CRISPR-Cas12a to the bubbles is closely related to the location and size of the bubble and the GC base content of crRNA. In addition, we used the unique property of CRISPR-Cas12a to invent a new method to detect mutations and successfully detect the CD41-42(-CTTT) mutation. The detection limit of this method is 0.001%. Overall, our results strongly indicate that in addition to considering off-target effects caused by base mismatches, a comprehensive off-target analysis of the insertion and deletion of the target dsDNA sequence is required, and specific guidelines for effectively reducing potential off-target cleavage are proposed, to improve the safety manual of CRISPR-Cas12a biological application.