Because DNA has the merit of high capacity and complexity, DNA steganography, which conceals DNA-encoded messages, is very promising in information storage. The classical DNA steganography method hides DNA with a "secret message" in a mount of junk DNA, and the message can be extracted by polymerase chain reaction (PCR) using specific primers (key), followed by DNA sequencing and sequence decoding. As leakage of the primer information may result in message insecurity, new methods are needed to better secure the DNA information. Here, we develop a pre-key by either mixing specific primers (real key) with nonspecific primers (fake key) or linking a real key with 3'-end redundant sequences. Then, the single-stranded DNA (ssDNA) trans cleavage activity of CRISPR/Cas12a is employed to cut a fake key or remove the 3'-end redundant sequences, generating a real key for further information extraction. Therefore, with the Cas12a-assisted DNA steganography method, both storage and transfer of DNA-encoding data can be better protected.
Keywords: CRISPR; Cas12a; DNA steganography; DNA storage; trans cleavage.